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PubMed2003

Detection of trisomy 21 by quantitative fluorescent-polymerase chain reaction in uncultured amniocytes.

Prenatal diagnosis of fetal trisomy 21 is usually performed by cytogenetic analysis. This requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. Today, trisomy 21 can be rapidly diagnosed within 24 h by molecular analysis of uncultured fetal cells using the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. The aim of our study was to test a chromosome quantification method on the basis of the analysis of fluorescent PCR products derived from non-polymorphic target genes. Co-amplification of a portion of DSCR1 (Down syndrome Critical Region 1) and the reference gene, CFTR (cystic fibrosis transmembrane regulator) enabled molecular detection of trisomy 21. Our method was successfully tested on a total of 154 amniotic fluids in a blind prospective study. Calculation of the DSCR1/CFTR ratio allowed us to distinguish between 152 normal amniotic fluids (mean ratio 0.99) and 2 amniotic fluids presenting a trisomy 21 status (DSCR1/CFTR ratio of 1.53 and 1.61, respectively). The results obtained by conventional cytogenetic analysis and our quantitative PCR method were concordant in every case. Our gene-based fluorescent PCR approach represents an alternative molecular method for rapid and reliable detection of trisomy 21, which can be helpful in the prenatal diagnosis of women at high risk of fetal trisomy 21.

Adult↗

PubMed1985

Slow exponential decay of rate of superoxide production in phorbol ester-activated human neutrophils.

Proper quantification of the superoxide (O2-) respiratory burst induced in human neutrophils is important for better understanding of the mechanism of action of stimulators and inhibitors. Reexamination of the reaction triggered by phorbol myristate acetate (PMA) indicated that it was a persistent process which lasted over 60 min. Plots of rates versus time show that rates of O2- release decayed logarithmically with a mean half-life (T1/2) of 21 +/- 6 min (SD), N = 12). Calculations of areas under curves indicate an average O2- yield of 217 +/- 99 nmol/10(6) cells. The inclusion of catalase in incubation mixtures did not alter the T1/2 or O2- yield, nor was the latter value affected by the quantitive scavenging of O2- by cytochrome c. Under certain conditions--the presence of excess dimethyl sulfoxide, the substitution of a less potent phorbol ester or activation of cells at high densities--the initial rate was either increased or decreased but a complementary alteration in the T1/2 resulted in little or no change in the total O2- yield. Retinol and retinol acetate decreased the initial rate, but retinoic acid enhanced it. By comparison, total O2- production was markedly reduced by all three agents with the following order of potency: retinoic acid greater than retinol greater than retinol acetate. In contrast, the serine protease inhibitor, TPCK, suppressed both the O2- yield and initial rate to a similar extent. On the basis of present observations, it is proposed that under normal conditions of PMA cellular activation, the logarithmic decay of the rate of O2- release was not due to autoinactivation of the O2--generating system, but rather to another factor, a possibility being the depletion of intracellular NADPH.

Humans↗

PubMed2003

Coexpression of calcitonin receptor-like receptor and receptor activity-modifying protein 2 or 3 mediates the antimigratory effect of adrenomedullin.

Three isoforms of the receptor activity-modifying protein (RAMP) are thought to transport the calcitonin receptor-like receptor (CRLR) to the plasma membrane to function as calcitonin gene-related peptide or adrenomedullin receptors, but their role remains largely unknown. We investigated whether coexpression of RAMP and CRLR are involved in the regulation of cell migration using a monolayer-wounding protocol. Quantification of gene transcripts revealed expression of all RAMP isoforms and CRLR in cultured rat vascular smooth muscle cells (VSMCs), RAMP2 and RAMP3 in rat endothelial cells, and RAMP1 in rat fibroblasts. CRLR expression was minimal in endothelial cells and fibroblasts. Adrenomedullin potently suppressed the migration of VSMCs, whereas calcitonin gene-related peptide did not suppress migration in any cell type. The antimigratory effect of adrenomedullin on VSMCs was potentiated by transfecting CRLR cDNA. Cotransfection of RAMP2 or RAMP3 with CRLR into VSMCs resulted in a slower migratory rate, and this effect was enhanced by adrenomedullin. Migration of fibroblasts was also suppressed after cotransfection of RAMP2 or RAMP3 with CRLR. cAMP agonists had no effect on VSMC migration, and a cAMP antagonist failed to abrogate the antimigratory effect of adrenomedullin. Thus, coexpression of CRLR and RAMP2 or RAMP3 mediates the inhibitory effect of adrenomedullin on cell migration, independent of cAMP-dependent signaling pathways.

Adrenomedullin↗

PubMed1991

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations.

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.

Cell Compartmentation↗

PubMed1995

Regulation of phosphatidylinositide transduction system in the rat spinal cord during aging.

Age-related functional alterations in a variety of neurotransmitter systems result in modulation of interneuronal communications which has some relevance in neurological deficits observed in the aging process. The synergistic interactions between protein kinase and inositol 1,4,5-trisphosphate (insP3)/Ca2+ pathways underlie a variety of cellular responses to external stimuli. To determine whether age-dependent changes occur in the regulation of protein kinase C and inositol 1,4,5-trisphosphate/Ca2+ pathways, insP3 contents as a marker for the release of intracellular calcium, saturation binding analysis of Ins P3 receptor using [3H]inositol 1,4,5-trisphosphate, slot/northern blot analysis of Ins P3 receptor-encoding mRNA transcripts, and the activities of Ca2+/phospholipid-dependent protein kinase C isozymes were investigated in the rat spinal cord. Inositol 1,4,5-trisphosphate content and [3H]inositol 1,4,5-trisphosphate binding site density (Bmax) were quantified in the spinal cords of young (three months old), adult (12 months old) and senescent (25 months old) male Fischer 344 rats. Spinal cord content of inositol 1,4,5-trisphosphate was increased (P < 0.01) in the 25-month old compared to the three- and 12-month old animals. The density of Ins P3 receptor in particulate membranes derived from the 25-month old rats was reduced (P < or = 0.01), but the binding affinity (Kd) was increased (P < or = 0.04) by a factor of 2.2 and 3.2 at 25 months of age when compared with three- and 12-month old animals, respectively. Young and middle-aged animals showed no differences in both inositol 1,4,5-trisphosphate contents and [3H]inositol 1,4,5-trisphosphate binding site density. The quantity of Ins P3 receptor mRNA was significantly increased with age in the order 25 >> 12 > 3 months of age. Total functional cytosolic and membrane-associated PKC activities were decreased (P < or = 0.05) in the 25-month compared to the three- and 12-month old rats in which activity remained unchanged. Total membrane/cytosolic activity ratios were unchanged by the aging process. In all cases, the activities of membrane-associated conventional protein kinase C isozymes (alpha, beta and gamma), determined by immunoprecipitation followed by in situ quantification of protein kinase C activities in the immunoprecipitates, showed age-dependent decline. The activities of protein kinase C-alpha and beta were significantly decreased in age-related manner. However, the activity of the gamma-isozyme was not significantly changed at 12- and 25-months of age, although it was higher (P < or = 0.03) in young rats. Western blot analyses using affinity purified polyclonal antibodies specific for each isozyme indicated a single protein with an apparent molecular mass of approximately 80 x 10(3) molec. weight for all isozymes except for the beta isozyme that also had an appreciable immunoreactive band at approximately 36 x 10(3) molec. weight. Overall, the aging process did not affect the electropheretic mobility of each isozyme. With decreased protein kinase C activity, the present data suggest that the aging process would decrease protein kinase C-induced phosphorylation of membrane proteins including Ins P3 receptor. A significant change in Ins P3 receptor affinity combined with increased levels of Ins P3 receptor mRNA-encoding transcripts in senescent rats suggests not only a modification (possibly by phosphorylation) of Ins P3 receptor protein but also the existence of multiple (spliced) variants of Ins P3 receptor in spinal neurons with increasing age. The present data indicate that the spinal contents of inositol 1,4,5-trisphosphate increased with age, but with decreased efficacy and number of inositol 1,4,5-trisphosphate-activatable Ca2+ channels in the spinal cord of senescent rats. These age-related changes may contribute to the attenuated responsiveness of spinal cord neurons by phosphoinositide-coupled receptors during the aging process.

Aging↗

PubMedDate not supplied

The effect of spinal instrumentation particulate wear debris. an in vivo rabbit model and applied clinical study of retrieved instrumentation cases.

STUDY DESIGN: The current study was undertaken to determine if the presence of spinal instrumentation wear particulate debris deleteriously influences early osseointegration of posterolateral bone graft or disrupts an established posterolateral fusion mass. OBJECTIVES: Using an in vivo animal model, the first phase (basic science) of this study was to evaluate the effect(s) of titanium wear particulate on a posterolateral spinal arthrodesis based on serological, histological and immunocytochemical analyses. The second phase (clinical) was to perform the same analysis of soft tissue surrounding spinal instrumentation in 12 symptomatic clinical patients. SUMMARY OF BACKGROUND DATA: The effect of unintended wear particulate resulting from micromotion between the interconnection mechanisms in spinal instrumentation remains a clinical concern. METHODS: Thirty-four New Zealand White rabbits were randomized into two groups based on postoperative time periods of 2 months (Group 1, n=14) and 4 months (Group II, n=20). Group I underwent a posterolateral arthrodesis (PLF) at L5-L6 using tricortical iliac autograft or tricortical iliac autograft plus titanium particulate. Group 2 all received iliac autograft at the initial surgery and were reoperated on after 8 weeks and treated with PLF exposure alone or titanium particulate. Postoperative analysis included serological quantification of systemic cytokines. Postmortem microradiographic, immunocytochemical and histopathological assessment of the intertransverse fusion mass quantified the extent of osteolysis, local proinflammatory cytokines, osteoclasts and inflammatory infiltrates. Clinical aspect of study: Over the last 2 years, 12 patients more than 0.4 years after spinal instrumentation presented with painful paraspinal inflammation. At surgical exploration, the cultures were negative for infection and the surrounding soft tissue was examined for cytokine reactions. There was loosening of implants and osteolysis in the location of the wear debris in 8 of 12 patients. RESULTS: Basic science phase: serological analysis of systemic cytokines indicated no significant differences in cytokine levels (p>.05) between the titanium or autograft treatments. Immunocytochemistry indicated increased levels of local cytokines: TNF-alpha at the titanium-treated PLF sites at both time periods (p<.05). Osteoclast cell counts and regions of osteolytic resorption lacunae were higher in the titanium-treated versus autograft-alone groups (p<.05), and the extent of cellular apoptosis was markedly higher in the titanium-treated sites at both time intervals. Electron microscopy indicated definitive evidence of phagocytized titanium particles and foci of local, chronic inflammatory changes in the titanium-treated sites. Clinical aspect: Eleven of 12 clinical cases demonstrated elevated TNF-alpha levels and an increased osteoclastic response in the vicinity of wear debris caused by dry frictional wear particles of titanium or stainless steel. Osteolysis most commonly involved loose transverse connectors. Resection of the wear debris and surrounding fibroinflammatory glycocalyx resulted in resolution of clinical symptoms in all 12 cases. CONCLUSIONS: Titanium particulate debris introduced at the level of a spinal arthrodesis elicits a cytokine-mediated particulate-induced response favoring proinflammatory infiltrates, increased expression of intracellular TNF-alpha, increased osteoclastic activity and cellular apoptosis. This is the first basic scientific study and the first clinical study demonstrating associations of spinal instrumentation particulates wear debris and increased cytokines and increased osteoclastic activity. Osteolysis is the number one cause of failure of orthopedic implants in the appendicular skeleton. Spinal surgeons need to increase their awareness of this destructive process.

Acid Phosphatase↗

PubMed2003

Orexin-A immunoreactivity and prepro-orexin mRNA expression in hyperphagic rats induced by hypothalamic lesions and lactation.

Orexins are endogenous neuropeptides that potently facilitate appetite and food consumption. In the present study, we examined orexin immunoreactivity and prepro-orexin mRNA expression in the lateral hypothalamus by immunohistochemistry and competitive reverse transcription-polymerase chain reaction (RT-PCR) methods in different models of hyperphagia in rats. Hyperphagia was induced by lesions of either the ventromedial hypothalamus (VMHL) or the paraventricular nucleus (PVNL), and we also compared lactating rats to nonlactating controls. Both VMHL and PVNL increased food intake and body weight compared to shams. On day 7 post lesion, serum leptin and insulin concentrations exhibited 3.2- and 2.8-fold increases in VMHL rats, and nonsignificant 1.8- and 1.8-fold increases in PVNL rats; there were significant decreases (48% and 33%) in lactating rats on day 12 postpartum compared to controls, respectively. Serum glucose concentrations were not significantly changed compared to controls in these rats. Quantification by image analysis suggests that VMHL significantly decreased the number and mean staining intensity of orexin-A immunoreactive neurones compared to those in the sham-lesioned group; while PVNL did not change orexin-A immunoreactivity. Competitive RT-PCR analysis showed that VMHL significantly decreased the prepro-orexin mRNA expression compared to those in the sham-lesioned group, and PVNL did not change it. Lactating rats on days 11-12 of lactation had significantly greater number and mean staining intensity of orexin-A immunoreactive neurones, prepro-orexin mRNA expression food intake and body weight than nonlactating postpartum rats. Thus, changes in orexin-A immunoreactivity and prepro-orexin mRNA expression were not consistent between the hyperphagia models. These results suggest that the hyperphagia from VMHL or PVNL and lactating rats differ in their involvement of orexin-A, and the change in circulating leptin and insulin concentrations may be involved in the change of orexin-A immunoreactivity in these rats.

Animals↗

PubMed1985

Cytochemistry of membrane proteases.

Membrane proteases that are detectable by cytochemical means are the classified exopeptidases, aminopeptidases A and M (or N), gamma-glutamyl transpeptidase (which also acts as transferase), dipeptidyl peptidase IV and the endopeptidase, enteropeptidase (also known as enterokinase). Not yet classified are the possible exopeptidase, tripeptidyl peptidase and endopeptidases I (Ala-endopeptidase) and II (Arg-endopeptidase). All these membrane proteases can be investigated with either chromogenic or fluorogenic procedures using synthetic peptide substrates. The most useful substrates are 4-methoxy-2-naphthylamine amino acids and peptides for cytochemical localizations at the light and electron microscope levels, for cytophotometric quantification and the study of membrane protease isoenzymes after analytical isoelectric focusing. Amino acid or peptide derivatives of naphthylamine AS can be recommended for light microscopical localization and cytofluorometric quantification, and 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin amino acids and peptides for the development of enzyme bands after isoelectric focusing. Cytochemistry reveals the heterogeneity in the distribution and species differences of membrane proteases in adult cells, tissues and organs and during development. It also reveals some common localizations, such as in small intestinal enterocytes and proximal tubule cells. The species and organ differences are substantiated and extended considerably by isoelectric focusing in combination with methods for the cytochemical detection of proteases. In addition, continuous cytophotometry or cytofluorometry (section and cultured cell biochemistry) allows the kinetic characteristics, initial reaction rates and maximum activities of all membrane proteases to be determined. The physiological functions of the endopeptidases and exopeptidases are still a matter of debate. However, from cytochemical inhibition studies with natural peptide substrates, e.g. peptide hormones, there is increasing evidence that the proteases detected with synthetic peptides play a decisive role in many physiological circumstances, e.g. in endocrine regulation mechanisms or the regulation of blood pressure. In this respect, capillary endothelium-linked surface membrane proteases may be especially important.

Animals↗

PubMed2004

Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilized plant mitochondria.

The inner mitochondrial membrane is selectively permeable, which limits the transport of solutes and metabolites across the membrane. This constitutes a problem when intramitochondrial enzymes are studied. The channel-forming antibiotic AlaM (alamethicin) was used as a potentially less invasive method to permeabilize mitochondria and study the highly branched electron-transport chain in potato tuber (Solanum tuberosum) and pea leaf (Pisum sativum) mitochondria. We show that AlaM permeabilized the inner membrane of plant mitochondria to NAD(P)H, allowing the quantification of internal NAD(P)H dehydrogenases as well as matrix enzymes in situ. AlaM was found to inhibit the electron-transport chain at the external Ca2+-dependent rotenone-insensitive NADH dehydrogenase and around complexes III and IV. Nevertheless, under optimal conditions, especially complex I-mediated NADH oxidation in AlaM-treated mitochondria was much higher than what has been previously measured by other techniques. Our results also show a difference in substrate specificities for complex I in mitochondria as compared with inside-out submitochondrial particles. AlaM facilitated the passage of cofactors to and from the mitochondrial matrix and allowed the determination of NAD+ requirements of malate oxidation in situ. In summary, we conclude that AlaM provides the best method for quantifying NADH dehydrogenase activities and that AlaM will prove to be an important method to study enzymes under conditions that resemble their native environment not only in plant mitochondria but also in other membrane-enclosed compartments, such as intact cells, chloroplasts and peroxisomes.

Alamethicin↗

PubMed1987

Protein 4.1 in sickle erythrocytes. Evidence for oxidative damage.

Sickle erythrocytes are known to undergo excessive auto-oxidation, resulting in the generation of increased intracellular levels of several species of free radical oxidants. This environment is likely to enhance the accumulation of oxidative lesions by membrane components, although, as yet, this has been shown directly only for the sickle membrane phospholipids. We examined the oxidative status of protein 4.1, a major component of the human erythrocyte protein skeleton. We found that protein 4.1 isolated from sickle erythrocytes bound approximately 4-fold less to protein 4.1-stripped membranes than did the normal protein. The binding defect was inherent in the sickle protein and not in its membrane-binding site(s) since normal protein 4.1 bound to sickle protein 4.1-stripped inside-out vesicles similar to normal protein 4.1-stripped inside-out vesicles. Sickle membranes, in particular spectrin-depleted inside-out vesicles, contained less protein 4.1 than normal membranes. Purified sickle protein 4.1 contained 20-40% high molecular weight aggregated protein (Mr greater than 200,000), whereas the purified normal protein contained approximately 10% high molecular weight protein. The high molecular weight protein was immunoreactive with antibodies to protein 4.1 but not with antibodies to spectrin, ankyrin, band 3, glycophorin, or hemoglobin, suggesting that the high molecular weight protein was cross-linked protein 4.1 and not a complex of protein 4.1 and some other membrane protein(s). Purified sickle protein 4.1 was eluted from an anion-exchange resin at a higher salt concentration than normal protein 4.1. Oxidizing normal protein 4.1 with diamide resulted in an anion-exchange elution pattern similar to the sickle protein, suggesting that oxidation can affect protein surface charge. Activated thiol beads bound one-half as much sickle protein 4.1 as normal protein 4.1 when both were solubilized directly from membranes, demonstrating that thiol oxidation had occurred in vivo. Direct quantification of protein thiols revealed that the sickle protein contained 1-2 mol% fewer cysteines/protein 4.1 monomer than did the normal protein. By amino acid analysis, sickle protein 4.1 was found to contain less methionine and tyrosine than did the normal protein and contained approximately 1 mol% cysteic acid, whereas the normal protein did not contain any cysteic acid. Taken together, our results strongly suggest that sickle protein 4.1 has sustained oxidative damage in vivo. This damage can alter the functional properties of the sickle protein and may be an underlying factor in the myriad of membrane abnormalities reported in sickle erythrocytes.

Anemia, Sickle Cell↗

PubMed2002

Quantification of the response of rat medullary raphe neurones to independent changes in pH(o) and P(CO2).

The medullary raphe nuclei contain putative central respiratory chemoreceptor neurones that are highly sensitive to acidosis. To define the primary stimulus for chemosensitivity in these neurones, the response to hypercapnic acidosis was quantified and compared with the response to independent changes in P(CO2) and extracellular pH (pH(o)). Neurones from the ventromedial medulla of neonatal rats (P0-P2) were dissociated and maintained in tissue culture for long enough to develop a mature response (up to 70 days). Perforated patch clamp recordings were used to record membrane potential and firing rate while changes were made in pH(o), P(CO2) and/or [NaHCO(3)](o) from baseline values of 7.4, 5 % and 26 mM, respectively. Hypercapnic acidosis (P(CO2) 9 %; pH(o) 7.17) induced an increase in firing rate to 285 % of control in one subset of neurones ('stimulated neurones') and induced a decrease in firing rate to 21 % of control in a different subset of neurones ('inhibited neurones'). Isocapnic acidosis (pH(o) 7.16; [NaHCO(3)](o) 15 mM) induced an increase in firing rate of stimulated neurones to 309 % of control, and a decrease in firing rate of inhibited neurones to 38 % of control. In a different group of neurones, isohydric hypercapnia (9 % P(CO2); [NaHCO(3)](o) 40 mM) induced an increase in firing rate of stimulated neurones by the same amount (to 384 % of control) as in response to hypercapnic acidosis (to 327 % of control). Inhibited neurones also responded to isohydric hypercapnia in the same way as they did to hypercapnic acidosis. In Hepes-buffered solution, both types of neurone responded to changes in pH(o) in the same way as they responded to changes in pH(o) in bicarbonate-buffered Ringer solution. It has previously been shown that all acidosis-stimulated neurones in the medullary raphe are immunoreactive for tryptophan hydroxylase (TpOH-ir). Here it was found that TpOH-ir neurones in the medullary raphe were immunoreactive for carbonic anhydrase type II and type IV (CA II and CA IV). However, CA immunoreactivity was also common in neurones of the hypoglossal motor nucleus, inferior olive, hippocampus and cerebellum, indicating that its presence is not uniquely associated with chemosensitive neurones. In addition, under the conditions used here, acetazolamide (100 microM) did not have a significant effect on the response to hypercapnic acidosis. We conclude that chemosensitivity of raphe neurones can occur independently of changes in pH(o), P(CO2) or bicarbonate. The results suggest that a change in intracellular pH (pH(i)) may be the primary stimulus for chemosensitivity in these putative central respiratory chemoreceptor neurones.

Acidosis↗

PubMed2006

Do extracellular matrix protein expressions change with cyclic reproductive hormones in pelvic connective tissue from women with stress urinary incontinence?

BACKGROUND: To evaluate differential expression of transforming growth factor (TGF-beta1), latent transforming factor-binding proteins (LTBP-1, LTBP-2) and elastin microfibril components (fibrillin-1 and fibrillin-2) in vaginal tissue from women with stress urinary incontinence (SUI). METHODS: In this case-control study, vaginal tissue from women in both phases of the menstrual cycle was obtained. Messenger RNA (mRNA) expressions of LTBP-1, LTBP-2, fibrillin-1, fibrillin-2 and TGF-beta1 were determined by relative real-time quantification PCR. Tissue localization was analysed by immunohistochemistry, and semiquantitative protein expression was evaluated by Western blot analysis. RESULTS: Vaginal wall fibroblasts synthesized all proteins tested. LTBP-1, LTBP-2 and TGF-beta1 co-localized with elastin microfibrils, fibrillin-1 and fibrillin-2 in the extracellular matrix. LTBP-1 mRNA and protein expressions were higher in control versus women affected with SUI in the proliferative phase (P = 0.04), while in the secretory phase, mRNA expression in cases was higher (P = 0.04). Fibrillin-1 mRNA was higher in women affected by SUI versus controls in both phases, but no statistical differences in fibrillin-1 protein expression were observed between the two groups in either phase. LTBP-2 and TGF-beta1 mRNA expressions showed the same trends as LTBP-1. CONCLUSION: LTBP-1, LTBP-2, TGF-beta1, fibrillin-1, and fibrillin-2 expressions are hormonally regulated in vaginal wall fibroblasts and differ in women affected by SUI when compared to controls. These data suggest a mechanism to regulate TGF-beta1 activity in pelvic connective tissue.

Case-Control Studies↗

PubMed2003

Spinal implant debris-induced osteolysis.

STUDY DESIGN: Generally, implant-induced osteolysis is a manifestation of an adverse cellular response to phagocytosable particulate wear and corrosion debris. Initially termed "cement disease," particle-induced loosening was recognized by Charnley in the early 1960s. Despite the plethora of information gained over the last 40 years on the basic science of periprosthetic bone loss, much remains unanswered. The effect of unintended debris resulting from wear and corrosion (e.g., micromotion between the interconnection mechanisms in spinal implants) remains a clinical concern. The current study highlights what is known of particle-induced osteolysis and how the presence of spinal implant particulate debris deleteriously influences osseointegration of posterolateral bone graft or disrupts an established posterolateral fusion mass. Tissue explant, animal, and cell culture studies have revealed the complexity of cellular reactivity involved in aseptic particle-induced osteolysis. OBJECTIVES: The objectives of this study are twofold: 1) to highlight the dominant cellular participants in total joint arthroplasty particle induced osteolysis, which are purportedly the macrophage, osteoblast, fibroblast, and osteoclast and several of the dominant chemical mediators have been identified as well, which include prostaglandin E2, tumor necrosis factor-alpha, interleukin-1, and interleukin-6; and 2) to demonstrate the potential deleterious effects of spinal implant debris using animal models and analysis of soft tissue surrounding spinal implants in symptomatic patients. METHODS: There are a growing number of proinflammatory and anti-inflammatory cytokines, prostenoids, and enzymes that have been shown to play important roles in the pathology of particle-induced osteolysis. Reports that aseptic granulomatous inflammation typical of that associated with corrosion debris appear to correlate with the complexity of the implant. Titanium particulate material was used to induce effects in 34 New Zealand White rabbits where analysis included serological quantification of systemic cytokines. Postmortem microradiographic, immunocytochemical, and histopathologic assessment of the intertransverse fusion mass quantified the extent of osteolysis, local proinflammatory cytokines, osteoclasts and inflammatory infiltrates. Clinical analysis of 12 patients more than 0.4 years after spinal implants (mean 4.03, range 0.4 to 11 years) presented with late operative site pain. RESULTS: Currently the etiology of this inflammation around spinal implants resembles particle-induced osteolysis around joint arthroplasties where there typically is a self-perpetuating fibroinflammatory zone adjacent to the implant, where macrophage exhaustion, reactive oxygen intermediates, and pro-inflammatory cytokines affect a host of local cell types and induce a widening zone of soft tissue damage and inflammation. Animal model analysis indicated increased levels of local inflammatory cytokines typically associated with osteolysis-tumor necrosis factor-alpha. Osteoclast cell counts and regions of osteolytic resorption lacunas were higher in the titanium-treated versus autograft-alone groups (P < 0.05), and the extent of cellular apoptosis was markedly higher in the titanium-treated sites at both time intervals. Electron microscopy indicated definitive evidence of phagocytized titanium particles and foci of local, chronic inflammatory changes in the titanium-treated sites. CLINICAL CASES: 11 of 12 clinical cases demonstrated elevated tumor necrosis factor-alpha levels and an increased osteoclastic response in the vicinity of wear debris caused by dry frictional wear particles of titanium or stainless steel. Resection of the wear debris and surrounding fibroinflammatory zone resolved clinical symptoms in all 12 cases. CONCLUSIONS: More basic science and clinical research is needed to develop novel strategies for gaining knowledge, and developing effective evaluation and treatment of patients with implant debris related osteolysis. Titanium debris simulating that produced by spinal implants introduced at the level of a spinal arthrodesis elicits an inflammatory cytokine mediated particulate-induced response through increased expression of intracellular TNF-alpha, increased osteoclastic activity and cellular apoptosis. This study highlighted the association between spinal implants particulate wear debris and increased potential for osteolysis. Aseptic osteolysis is among the primary reasons for failure of orthopedic implants. Increased awareness of this destructive process is becoming more important with the growing popularity of total disc arthroplasty and highly modular spinal implants.

Adult↗

PubMed2002

Real-time quantitative RT-PCR assay of GADD45 gene expression changes as a biomarker for radiation biodosimetry.

PURPOSE: To assess the efficacy of fluorescent-based quantitative reverse transcription-polymerase chain reaction (QRT-PCR) technology to measure gene expression changes (GEC) for rapid, point-of-care radiation dose assessment. MATERIALS AND METHODS: A real-time QRT-PCR assay based on 5'-fluorogenic nuclease TaqMan(TM) methodology was developed, which employs both relative and absolute quantification of a candidate mRNA biomarker. Growth arrest and DNA damage gene 45 (GADD45), a cell-cycle regulation and DNA repair gene, served as the paradigm because of the reported linear dose-response relationship for mRNA induction in the human myeloid tumor cell line (ML-1) over the range of 2-50 cGy. Using an ex vivo whole-blood model, GEC was measured from total blood RNA at 24h and 48 h after (60)Co gamma-ray exposures (0-3 Gy; 0.1 Gy/min). RESULTS: A linear and reproducible up-regulation representing a twofold to fourfold change in GADD45 relative and absolute GEC was confirmed in both intra- and inter-assay analyses. CONCLUSIONS: Primer and probes to detect GADD45 targets using real-time PCR were developed. This is the first report using realtime QRT-PCR to measure radiation-induced GEC dose response. Real-time QRT-PCR using GEC as biomarkers offers rapidity, sensitivity, and reproducibility as a potential efficient biological dosimetry tool applicable in radiation therapy applications and early-response accident biodosimetry.

Biomarkers↗

PubMed1985

Changes in intra- and extrahepatic VLDL in the rat following acute injury by thioacetamide. A morphometric and biochemical study.

The effect of a single dose of TAA (100 mg/kg body weight) on intra- and extrahepatic lipoproteins of the very low density (VLDL) type was studied in rats by morphometric and biochemical methods. For a better quantification of VLDL in the periportal (zone 1) and centrilobular (zone 3) hepatocytes of control and TAA-intoxicated livers, colchicine was used as an inhibitor of hepatic lipoprotein secretion. Generally, there exists a great functional heterogeneity between the hepatocytes of zone 1 and 3 in the colchicine-treated controls manifested in a significantly different accumulation rate of VLDL particles. The mean number of VLDL particles per vesicle, the mean secretory vesicle size and the volume density of the electron-lucent secretory vesicles are two times larger in hepatocytes of zone 1 than zone 3. The volume of the VLDL particles amounts to 7.3 X 10(-5) microns3 and 22 X 10(-5) microns3 in the peri- and centrilobular regions. On the other hand, there is no significant lobular-zonal difference in the number of light and dark secretory vesicles. Within 48 h TAA treatment causes a reduction in the number of VLDL particles/100 microns 2 in zone 1 and 3 by 66% and 61%, whereas the number of light secretory vesicles is decreased by 31% and 58%, respectively. The volume density of the latter is significantly diminished only in zone 1. Moreover, the VLDL particle volume is reduced to nearly 50% in each lobular zone examined. The data obtained after TAA treatment from the electron-dense secretory vesicles do not differ significantly from those of the colchicine-treated controls. Acute TAA intoxication lowers the hepatic VLDL-TG output by about 50% in comparison with controls. The steady state of the serum TG concentration after TAA application implies that the clearance of TG from the serum must be diminished to the same extent as the hepatic TG output is found to decrease due to acute liver injury. The results presented here support our view that acute TAA intoxication lowers the hepatic VLDL output by inhibiting the intracellular formation of VLDL. The intrahepatic degradation of the newly synthesized VLDL seems to be unaffected. Despite the fact that the substructure of the hepatocytes in zone 3 is much more changed than in zone 1 after TAA treatment the quantitative data on the VLDL secretory products provide evidence that the process of lipoprotein formation is disturbed to nearly the same extent by TAA both in zone 1 and 3.

Acetamides↗

PubMed1990

A novel antioxidant role for hemoglobin. The comproportionation of ferrylhemoglobin with oxyhemoglobin.

Ferrylhemoglobin (X-FeIV-OH, where X denotes an amino acid residue in the globin moiety) has long been suspected as a cytotoxic agent produced by the interaction of oxyhemoglobin (X-FeIIO2) or methemoglobin (X-FeIII) with H2O2 in red blood cells. To date, however, technical difficulties have prevented the identification and quantification of X-FeIV-OH. Oxyhemoglobin exposed to a continuous flux of H2O2 (generated at a rate of 120 microM/min during the glucose oxidase-catalyzed oxidation of glucose) was oxidized to (a) X-FeIV-OH when [X-FeIIO2] less than 75 microM and (b) X-FeIII when [X-FeIIO2] greater than 75 microM (the production of X-FeIII proceeded with intermediate formation of X-FeIV-OH). The reduction of the X-FeIV-OH to X-FeIII could be explained by either of two alternative mechanisms: a O2(-)-mediated X-FeIV-OH---X-FeIII transition or a comproportionation of X-FeIV-OH and X-FeIIO2 to yield X-FeIII (a process mediated by a tyrosine moiety in the hemoprotein). The low rate of X-FeIIO2 autoxidation plus the negligible decrease in the rate of X-FeIII formation in the presence of either native or heat-denatured superoxide dismutase or apoenzyme (1 microM) suggested that O2- does not contribute to the reduction of X-FeIV-OH. Moreover, the dependence of X-FeIII formation on X-FeIIO2 concentration, together with the results of O2 uptake and H2O2 consumption measurements, provide experimental evidence to support the comproportionation reaction. Comproportionation is apparently catalyzed by intermolecular electron transfer between tyrosine residues, since the reaction did not occur when tyrosine residues were blocked by acetylation. Intact red blood cells exposed to the same flow rate of H2O2 presented a spectral profile which could be explained as a transition from X-FeIIO2 to X-FeIII. The intermediate production of X-FeIV-OH was detected by adding Na2S (2 mM), which revealed a spectral profile identical with that obtained with purified X-FeIV-OH. Measurements of concentrations and relative rate constants for the reaction of various intracellular reductants (glutathione, NAD(P)H, uric acid, ascorbic acid) with X-FeIV-OH revealed that comproportionation of X-FeIV-OH with X-FeIIO2 is the favored reaction. Our results provide (to our knowledge) the first definitive evidence for X-FeIV-OH in intact red blood cells. The rapid comproportionation reaction between X-FeIV-OH and X-FeIIO2 (to produce X-FeIII) explains why X-FeIV-OH has been elusive to date.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylation↗

PubMed2001

Detection and quantification of protein phosphatase inhibitor-1 gene expression in total rat liver and isolated hepatocytes.

The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrated using highly sensitive semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Quantification by real-time RT-PCR (LightCycler technology) yielded the same copy number of inhibitor-1 mRNA in total rat liver and isolated hepatocytes (12 copies per cell). This novel finding shows that rat liver expresses indeed inhibitor-1 mRNA, albeit in low amounts. The low copy number explains why the mRNA had not been detected by Northern blotting so far. For comparison, about 425 copies/cell were detected in brain and 2500 copies/cell in skeletal muscle from rat. The full-length coding sequence of rat liver inhibitor-1 was cloned and sequenced, 100% homology with the muscle cDNA was obtained, indicating the expression of the same gene in liver and muscle. In vitro transcription and translation yielded a protein (Mr approximately 30 kDa) which could be detected with a specific antibody by immunoblotting. This indicates an intact open reading frame of inhibitor-1 in rat liver. Immunoblotting of liver extract yielded a very weak band which comigrated with the inhibitor-1 proteins from muscle and brain. It is concluded that mRNA expression of inhibitor-1 may have implications for the regulation of protein phosphatase-1 (PP1) in rat liver.

Animals↗

PubMed1993

Reproducibility of lysis-centrifugation cultures for quantification of Mycobacterium avium complex bacteremia.

While quantitative mycobacterial blood cultures have been accepted as the standard for evaluating response to various Mycobacterium avium complex (MAC) treatment regimens, variability in this methodology has not been evaluated in a rigorous fashion. We thus studied the reproducibility of quantitative MAC cultures by a lysis-centrifugation culture system within and among five institutions. To measure the intralaboratory variation in mycobacterial colony counts, colony counts from duplicate blood specimens collected from 52 AIDS patients with MAC bacteremia were determined. Colony counts ranged from 0 to 50,000 CFU/ml. Nonparametric analyses revealed there was no significant difference in colony counts between the 52 duplicate specimens. The agreement between the intralaboratory paired specimens, as measured by the intraclass correlation coefficient, was 0.997. To measure the interlaboratory variation, multiple 10-ml aliquots from 12 patients were distributed to five institutions and processed within 24 to 32 h by lysis-centrifugation. For the 12 specimens distributed to the five laboratories, two-way analysis of variance for repeated measures revealed no significant difference in an individual patient's colony counts between laboratories (P > 0.2). We conclude that quantitation of mycobacterial colony counts by the lysis-centrifugation system is reproducible within and between institutions. Clinical trials evaluating response to therapeutic interventions for MAC can use multiple laboratories for quantitation of mycobacteremia. Furthermore, a 24- to 32-h delay in processing appeared to have no impact on reproducibility.

AIDS-Related Opportunistic Infections↗