SEARCH · PubMed Health

Results for “Intracellular quantification”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 703 records · Page 39Linked to original sources

PubMed2002

Deficiency of neuronal nitric oxide synthase (nNOS) worsens alcohol-induced microencephaly and neuronal loss in developing mice.

Previous work conducted in vitro suggests that nitric oxide (NO) protects developing neurons against the toxic effects of alcohol. We tested the hypothesis that neonatal mice carrying a null mutation for neuronal nitric oxide synthase (nNOS), the enzyme which synthesizes NO in neurons, have increased vulnerability to alcohol-induced microencephaly and neuronal loss. Wild-type mice and mutant (nNOS(-/-)) mice received a single intraperitoneal injection of ethanol (0.0, 2.2, 3.3, or 4.4 g/kg) daily over postnatal days (PD) 4-9 and were sacrificed on PD 10. Peak blood alcohol concentrations were approximately 170, 280, and 385 mg/dl for the 2.2, 3.3 and 4.4 g/kg/day treatment groups, respectively, and did not differ significantly between wild-type and nNOS(-/-) strains. Exposure to alcohol induced dose-dependent reductions in total brain weight, forebrain weight and cerebellum weight in both strains of mice. However, the reductions in brain weight were significantly more severe in the nNOS(-/-) mice than in wild type. Quantification of cerebellar neurons revealed that alcohol-induced losses of Purkinje cells and granule cells were both significantly greater in the nNOS(-/-) mice than in wild type. The increased vulnerability of nNOS-deficient neurons to alcohol-induced cell death was confirmed in vitro. Cerebellar granule cell cultures derived from nNOS(-/-) and wild-type mice were exposed for 24 h to 0, 100, 200 or 400 mg/dl ethanol. At each alcohol concentration, the nNOS(-/-) neurons had a significantly greater cell loss than did the wild-type neurons. The results demonstrate that deficiency of nNOS decreases the ability of developing neurons to survive the toxic effects of alcohol. Because NO upregulates intracellular cGMP, which can activate cGMP-dependent protein kinase (PKG), we hypothesize that the NO-cGMP-PKG pathway has a neuroprotective role against alcohol toxicity within the developing brain.

Animals↗

PubMed2005

Intracellular inhibitory effects of Velcade correlate with morphoproteomic expression of phosphorylated-nuclear factor-kappaB and p53 in breast cancer cell lines.

Velcade, a proteasome inhibitor, has been shown to inhibit DNA binding activity of nuclear factor-kappaB (NF-kappaB) and to stabilize p53 in vitro. But its impact, in the context of activated (phosphorylated and translocated) NF-kappaB and the expression of p53, has not been studied in breast cancer. It would be desirable to determine whether or not the immunohistochemical (IHC) expressions of activated NF-kappaB and of p53 can predict the effects of Velcade in viable tumor cells. To answer these questions, we selected 3 breast cancer cell lines (SKBR-3, MDA-175, and MDA-231), which are negative for hormonal receptors, but differ in HER-2/neu expression (strong, mild, and minimal, respectively). The 3 cell lines showed different expressions of phosphorylated (p)- NF-kappaB and p53, as evaluated using immunohistochemistry with visual quantification by brightfield microscopy. After being treated with Velcade for 2 days, MDA-231 cells showed markedly reduced proliferation, followed by SKBR-3 cells, and then by MDA-175 cells. There was strong correlation between the nuclear expression of either p-NF-kappaB or p53 and the inhibitory rate of Velcade in the 3 cell lines (r = 0.987 and 0.807, respectively). Western blotting showed an increase in inhibitor-kappaB (I-kappaB) expression in nuclei of MDA-231 and SKBR-3 cells, but not in MDA-175 cells, following exposure to Velcade. Velcade treatment resulted in cleaved caspase-3 expression in MDA-231 cells and in the overexpression of p53 and p21WAF1 in all 3 cell lines, as evaluated using Western blotting. In summary, morphoproteomic analysis of p-NF-kappaB and p53 can be correlated with the inhibitory effect of Velcade in vitro. We propose that this proliferative inhibition is variably associated with blocking p-NF-kappaB function by upregulation of nuclear I-kappaB, stabilization of p53, and induction of p21WAF1.

Antineoplastic Agents↗

PubMed1999

Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant aspergillus oryzae strain

Two alpha-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the alpha-amylase gene, were characterized with respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition, and metabolic fluxes through the central metabolism during glucose-limited chemostat cultivations. Citrate synthase and isocitrate dehydrogenase (NAD) activities were found only in the mitochondria, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase (NADP) activities were found only in the cytosol, and isocitrate dehydrogenase (NADP), glutamate oxaloacetate transaminase, malate dehydrogenase, and glutamate dehydrogenase (NAD) activities were found in both the mitochondria and the cytosol. The measured biomass components and ash could account for 95% (wt/wt) of the biomass. The protein and RNA contents increased linearly with increasing specific growth rate, but the carbohydrate and chitin contents decreased. A metabolic model consisting of 69 fluxes and 59 intracellular metabolites was used to calculate the metabolic fluxes through the central metabolism at several specific growth rates, with ammonia or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain.

Journal Article↗

PubMed2003

Influence of zinc deficiency on the mRNA expression of zinc transporters in adult rats.

The accumulation of zinc in the cell is a sum of influx and efflux processes via transporter proteins, like the four Zn transporters (ZnT1-4), the divalent cation transporter 1 (DCT1) and of storage processes mainly bound to metallothionein (MT). To study the effect of Zn deficiency on mRNA expression levels, adult rats were used as an animal model. Food intake was restricted to 8 g/day containing 2 microg Zn/g fortified with pure phytate in Zn deficiency rats and 58 microg Zn/g in controls (n = 7). At day 1, 2, 4, 7, 11, 16, 22, and 29 of Zn deficiency, 3 animals were sacrificed, respectively (n = 24). Zn deficiency was evident from reduced plasma Zn, plasma alkaline phosphatase activity and severe mobilization of Zn from tissue stores (mainly skeleton), while food intake and body weight remained unaffected. Tissues representing Zn absorption (jejunum, colon), Zn storage and utilization (muscle, liver), and Zn excretion (kidney) were retrieved. Total RNA contents increased in colon (p = 0.003) and trend to decrease in liver (p = 0.086). Zn deficiency was without effect on tissue total RNA concentrations in muscle tissue and kidney. Real-time reverse transcription (RT) polymerase chain reaction (PCR) assays were developed and a relative quantification on the basis of GAPDH was applied. Assays allowed a relative and accurate quantification of mRNA molecules with a sufficiently high sensitivity and repeatability. All known Zn transporter subtypes were found in the tissues. ZnT3 was newly elucidated and sequenced in rat tissues. Expression patterns and reactions to Zn deficiency were specific for the tissue analysed. Expression results imply that some transporters are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis. The most distinct changes of expression levels were shown in colon which can therefore be postulated as a highly Zn sensitive tissue. MT was down-regulated in all tissues, massively in liver (p < 0.001) and in colon (p = 0.002) and in tendency also in the jejunum and kidney. In parallel with intracellular Zn status it is a potent candidate gene for Zn deficiency. ZnT1 and ZnT2 showed a significant up-regulation of mRNA expression in colon (p = 0.032 and p = 0.026) and for ZnT2 a trend of down regulation in jejunum (p = 0.098). This study provides the first comparative view of regulation of gene expression and fully quantitative expression analysis of all known Zn transporters in a non growing adult rat model on a constant platform and therefore allows a direct comparison.

Animals↗

PubMed2004

Cytoskeletal genes regulation by chronic morphine treatment in rat striatum.

It has been previously suggested that morphine can regulate the expression and function of some proteins of the cytoskeleton. In the present study, we used real-time quantitative polymerase chain reaction to examine the effects of chronic morphine administration, in rat striatum, on 14 proteins involved in microtubule polymerization and stabilization, intracellular trafficking, and serving as markers of neuronal growth and degeneration. Chronic morphine treatment led to modulation of the mRNA level of seven of the 14 genes tested. Glial fibrillary acidic protein (Gfap) and activity-regulated cytoskeleton-associated protein (Arc) mRNA were upregulated, while growth associated protein (Gap43), clathrin heavy chain (Cltc), alpha-tubulin, Tau, and stathmin were downregulated. In order to determine if the regulation of an mRNA correlates with a modulation of the expression of the corresponding protein, immunoblot analyses were performed. With the exception of Gap43, the levels of Cltc, Gfap, Tau, stathmin, and alpha-tubulin proteins were found to be in good agreement with those from mRNA quantification. These results demonstrate that neuroadaptation to chronic morphine administration in rat striatum implies modifications of the expression pattern of several genes and proteins of the cytoskeleton and cytoskeleton-associated components.

Animals↗

PubMed2006

Horizontal cell receptive fields are reduced in connexin57-deficient mice.

Horizontal cells are coupled by gap junctions; the extensive coupling of the horizontal cells is reflected in their large receptive fields, which extend far beyond the dendritic arbor of the individual cell. In the mouse retina, horizontal cells express connexin57 (Cx57). Tracer coupling of horizontal cells is impaired in Cx57-deficient mice, which suggests that the receptive fields of Cx57-deficient horizontal cells might be similarly reduced. To test this hypothesis we measured the receptive fields of horizontal cells from wildtype and Cx57-deficient mice. First, we examined the synaptic connections between horizontal cells and photoreceptors: no major morphological alterations were found. Moreover, horizontal cell spacing and dendritic field size were unaffected by Cx57 deletion. We used intracellular recordings to characterize horizontal cell receptive fields. Length constants were computed for each cell using the cell's responses to concentric light spots of increasing diameter. The length constant was dependent on the intensity of the stimulus: increasing stimulus intensity reduced the length constant. Deletion of Cx57 significantly reduced horizontal cell receptive field size. Dark resting potentials were strongly depolarized and response amplitudes reduced in Cx57-deficient horizontal cells compared to the wildtype, suggesting an altered input resistance. This was confirmed by patch-clamp recordings from dissociated horizontal cells; mean input resistance of Cx57-deficient horizontal cells was 27% lower than that of wildtype cells. These data thus provide the first quantification of mouse horizontal cell receptive field size and confirm the unique role of Cx57 in horizontal cell coupling and physiology.

Animals↗

PubMed1984

[Significance of morphological findings in congestive cardiomyopathy].

In 48 consecutive patients with myocardial disease, left ventricular biopsies were taken during diagnostic heart catheterization. Morphometric quantification of ultrastructural alterations of the heart muscle was performed. The patients were subdivided into 3 groups by means of clinical criteria: Group A: 18 surviving patients with pathological ECG, normal left ventricular ejection fraction, and normal coronary arteries. The mean follow-up in this group was 22 months. Group B: 23 surviving patients with congestive cardiomyopathy, i.e., normal coronary arteries and depressed left ventricular ejection fraction. The mean follow-up in this group was 23 months. Group C: 7 patients with congestive cardiomyopathy, who died during the study period from cardiac complications. The mean survival time of these patients after heart catheterization was 23 months. Myocardial fiber diameter, volume fraction of interstitial fibrosis, intracellular volume fraction of myofibrils, and the total myofibrillar mass per 100 g heart muscle were determined by light microscopic morphometry. In a pilot study comprising 26 patients the reproducibility of the morphological results were assessed by investigation of two different biopsy specimens taken from different areas of the left ventricular wall of each patient. The study revealed a coefficient of variation of 6% for myocardial fiber diameter, 28% for myocardial fibrosis, and 4% for myofibrillar volume fraction. Since there are only poor results from morphological investigations of the diseased myocardium, it was the purpose of the follow-up study presented in this paper: To quantify ultrastructural alterations of the heart in congestive cardiomyopathy by morphometric techniques. To correlate the quantitative morphometric data with the contractile state of the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2001

Humoral coding and decoding.

Humoral communication systems are dynamically regulated. Most hormones are released in a pulsatile or burst-like manner into the bloodstream. It is well known that information coded in the frequency and amplitude of secretory pulses allows for the differential regulation of specific target cell function and structure. However, despite intensive study of transmembrane signalling relatively little is known about how the temporal dynamics of extracellular humoral stimuli specifically regulates the temporal pattern of intracellular signalling pathways, such as Ca2+-dependent signalling. Repetitive spikes of Ca2+ encode this information in their amplitude, duration and frequency, and are in turn decoded into the pattern of gene expression and phosphorylation of target proteins. Using a mathematical model for G protein-coupled Ca2+ signalling and information-theoretic approaches to stimulus reconstruction we have systematically quantified the amount of information coded in the Ca2+-signal about the dynamics of the stimulus, which allows us to explore the temporal bandwidth of transmembrane signalling. These in silico approaches permit us to differentiate the amount of information coded in the frequency, temporal precision, amplitude and the complete Ca2+-signal. This may open an avenue to the quantification of information flow and processing in the intra- and intercellular coding and decoding machinery.

Action Potentials↗

PubMed2006

Simulation analysis of interference EMG during fatiguing voluntary contractions. Part II--changes in amplitude and spectral characteristics.

Capabilities of amplitude and spectral methods for information extraction from interference EMG signals were assessed through simulation and preliminary experiment. Muscle was composed of 4 types of motor units (MUs). Different hypotheses on changes in firing frequency of individual MUs, intracellular action potential (IAP) and muscle fibre propagation velocity (MFPV) during fatigue were analyzed. It was found that changes in amplitude characteristics of interference signals (root mean square, RMS, or integrated rectified value, IEMG) detected by intramuscular and surface electrodes differed. RMS and IEMG of surface detected interference signals could increase even under MU firing rate reduction and without MU synchronisation. IAP profile lengthening can affect amplitude characteristics more significantly than MU firing frequency. Thus, an increase of interference EMG amplitude is unreliable to reflect changes in the neural drive. The ratio between EMG amplitude and contraction response can hardly characterise the so-called 'neuromuscular efficiency'. The recently proposed spectral fatigue indices can be used for quantification of interference EMG signals. The indices are practically insensitive to MU firing frequency. IAP profile lengthening and decrease in MFPV enhanced the index value, while recruitment of fast fatigable MUs reduced it. Sensitivity of the indices was higher than that of indices traditionally used.

Computer Simulation↗

PubMed2002

Rapid translocation of polarized MDCK cell monolayers by Leptospira interrogans, an invasive but nonintracellular pathogen.

Pathogenic spirochetes of the genus Leptospira are a major cause of human zoonotic infectious disease worldwide. After gaining entry through the skin, the organism causes disease by hematogenously disseminating to multiple organs. The mechanism by which it penetrates the mammalian cell barriers to disseminate is not well understood. In this study, we used a low-passage-number isolate of Leptospira interrogans to elucidate the invasive potential of this spirochete. Quantification of bacteria by dark-field microscopy revealed that pathogenic spirochetes were able to translocate through polarized MDCK cell monolayers at a rate significantly greater than that of nonpathogenic Leptospira or a recognized invasive bacterial pathogen, Salmonella: In contrast to Salmonella, L. interrogans did not alter transepithelial electrical resistance during cell translocation. Both transmission and scanning electron microscopy revealed tight association of the extracellular spirochetes with the host cell plasma membrane, without membrane perturbations suggestive of cytoskeletal rearrangement. Spirochetes were not observed within intercellular junctions or membrane-bound compartments inside cells. They were found within the cytoplasm of only 8% of the counted cells. These results indicate that Leptospira is an invasive but not a facultative intracellular organism. We propose that the rapid translocation of mammalian cells by pathogenic Leptospira is a mechanism designed to evade killing by host cells that permits the organism to quickly reach the bloodstream and disseminate to multiple organs.

Animals↗

PubMed1985

A comparison of subcellular element concentrations in frozen-dried, plastic-embedded, dry-cut sections and frozen-dried cryosections.

Biological X-ray microanalysis of diffusible elements within cellular and subcellular compartments requires preparation methods to retain electrolytes in the compartments they occupied in vivo. X-ray microanalysis of frozen-dried, plastic-embedded samples has been used to quantitate electrolytes at the cellular level. We have compared the subcellular elemental distribution in dry cut sections from such samples with that in ultrathin frozen-dried cryosections. Rat pancreases were quench-frozen onto a helium-vapor-cooled copper block. Cryosections were cut at 130-150 K, transferred using a Gatan cold stage, frozen-dried in the column and analysed at 190 K. Tissue fragments were frozen-dried at 190 K, and cut on a dry knife at 293 K. Both samples provided images permitting unambiguous identification of all major compartments except the Golgi complex. Intracellular potassium-to-sodium ratios obtained on frozen-dried plastic-embedded sections were lower than for cryosections (e.g. 1.77 in basal cytoplasm in plastic sections as compared to 4.34 for cryosections) and varied with the pre-embedding procedure (e.g. 1.77 in formaldehyde-fixed as compared to 2.87 in osmium-fixed plastic sections). Potassium gradients between adjacent organelles were large in cryosections and insignificant in plastic-embedded material. Higher cytoplasmic phosphorus, potassium and sulfur concentrations were observed in cryosections. Therefore, a redistribution of electrolytes and covalently bound elements occurred subcellularly in the plastic sections. Calcium was quantifiable in most organelles in cryosections but the plastic lowered sensitivity too much to permit routine calcium quantification. We conclude that in our hands frozen-dried, plastic-embedded samples were compromised and provided lower sensitivity than cryosections.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Interactions between the sorbitol pathway, non-enzymatic glycation, and diabetic vascular dysfunction.

BACKGROUND: Many lines of evidence attest to a multifactorial pathogenesis of diabetic complications in humans and in animal models of diabetes. Increased sorbitol pathway metabolism and non-enzymatic glycation products have been implicated by many investigators in the pathogenesis of vascular and neural dysfunction as well as early vascular structural changes in animal models of diabetes. The present studies were undertaken to assess the mechanisms that mediate vascular dysfunction associated with these biochemical imbalances. METHODS: Three different animal models of diabetes were used: (1) rats with diabetes induced by injection of streptozotocin; (2) non-diabetic rats with acute hyperglycaemia of 5 h duration induced by i.v. glucose infusion at a rate sufficient to produce plasma glucose levels comparable to those in diabetic rats; and (3) the skin chamber granulation tissue model in which vessels in the chamber are exposed to buffer containing 5 or 30 mM glucose +/- pharmacological agents or 0.1 microM glycated rat serum albumin +/- pharmacological agents. Vascular function was assessed by injection of 11.3 microns 46Sc microspheres for quantification of blood flow and by injection of [125I] and [131I]bovine serum albumin for quantification of vascular albumin permeation. RESULTS: Vascular dysfunction induced by elevated glucose levels (increased blood flow and increased albumin permeation) in all three models was prevented by inhibitors of sorbitol pathway metabolism, inhibitors of nitric oxide synthesis and inhibitors of prostaglandin synthesis. In the skin chamber model vascular dysfunction induced by elevated glucose levels and by glycated rat serum albumin was prevented by superoxide dismutase, probucol and inhibitors of nitric oxide synthase. CONCLUSIONS: These observations suggest that vascular dysfunction induced by increased sorbitol pathway metabolism (caused by elevated glucose levels) and by products of non-enzymatic glycation (at normal glucose levels) is mediated by a common final pathway consistent with a scenario in which: increases superoxide production-->increases intracellular calcium levels-->increases nitric oxide synthesis-->increases blood flow and increases vascular permeability.

Animals↗

PubMed1992

Autoradiographic analysis of the distribution of 125I-tyramine-cellobiose-LDL in atherosclerotic lesions of the WHHL rabbit.

It is well established that plasma lipoproteins enter the artery wall and play a role in the atherogenic process. However, it is still unclear where within developing atherosclerotic lesions lipoproteins accumulate and which arterial cells participate in the metabolism of these lipoproteins. For this reason, light and electron microscopic autoradiograms were prepared from sections of lesioned aortas of Watanabe heritable hyperlipidemic (WHHL) rabbits 44 hours after injection of 125I-tyramine cellobiose-low density lipoprotein (TC-LDL). After uptake of 125I-TC-LDL and intracellular degradation of the LDL protein, the nondegradable TC ligand remains trapped and thus demarcates the cells participating in the degradation of LDL. Results of other studies indicate that 48 hours after injection into WHHL rabbits, about one half of the 125I label present in lesions represents accumulated degradation products while the remaining 125I label is present as intact 125I-TC-LDL. The distribution of autoradiographic silver grains was analyzed at low resolution in fatty streaks, transitional lesions, and advanced atheroma. In all cases, the majority of silver grains were associated with superficially located subendothelial macrophage-derived foam cells. In more advanced lesions, labeling was predominant in foam cells situated within the lateral margins of the lesions. Morphometric quantification of the distribution of silver grains in electron photomicrographs of fatty streaks from two young WHHL rabbits strongly supported the data obtained at the light microscopic level. In early fatty streaks from the aortic arch and the thoracic and abdominal aortas, subendothelial macrophage-derived foam cells contained a high proportion of the silver grains (40-60% of the total) and accounted for between 30% and 40% of the lesion volume. In contrast, smooth muscle cells in the lesions contained only 7-10% of the total silver grains and accounted for approximately 20% of the lesion volume. Endothelial cells contained the most silver grains on a per-unit-volume basis by occupying only 1-2% of the lesion volume. However, the endothelium contained less than 5% of the total grains in lesions. The remaining silver grains (25-45%) were associated with the extracellular matrix, which constituted between 40% and 50% of the lesion volume. These data indicate that in the WHHL rabbit, subendothelial macrophage-derived foam cells avidly accumulate and metabolize LDL despite having few functional LDL receptors.

Animals↗

PubMed2006

Preclinical analyses of the therapeutic potential of allopregnanolone to promote neurogenesis in vitro and in vivo in transgenic mouse model of Alzheimer's disease.

Herein, we present data to support a preclinical proof of concept for the therapeutic potential of allopregnanolone to promote neurogenesis. Our recent work has demonstrated that the neuroactive progesterone metabolite, allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), (APalpha) induced, in a dose dependent manner, a significant increase in proliferation of neuroprogenitor cells (NPCs) derived from the rat hippocampus and human neural stem cells (hNSM) derived from the cerebral cortex [1]. Proliferative efficacy was determined by incorporation of BrdU and (3)H-thymidine, FACS analysis of MuLV-GFP-labeled mitotic NPCs and quantification of total cell number. Allopregnanolone-induced proliferation was isomer and steroid specific, in that the stereoisomer 3beta-hydroxy-5beta-pregnan-20-one and related steroids did not increase (3)H-thymidine uptake. Immunofluorescent analyses for the NPC markers, nestin and Tuj1, indicated that newly formed cells were of neuronal lineage. Furthermore, microarray analysis of cell cycle genes and real time RT-PCR and western blot validation revealed that allopregnanolone increased the expression of genes which promote mitosis and inhibited the expression of genes that repress cell proliferation. Allopregnanolone-induced proliferation was antagonized by the voltage gated L-type calcium channel blocker nifedipine consistent with the finding that allopregnanolone induces a rapid increase in intracellular calcium in hippocampal neurons via a GABA type A receptor activated L-type calcium channel. Preliminary in vivo data indicate that APalpha for 24 hrs significantly increased neurogenesis in dentate gyrus, as determined by unbiased stereological analysis of BrdU positive cells, of 3-month-old male triple transgenic Alzheimer's disease mice. The in vitro and in vivo neurogenic properties of APalpha coupled with a low molecular weight, easy penetration of the blood brain barrier and lack of toxicity, are key elements required for developing APalpha as a neurogenic / regenerative therapeutic for restoration of neurons in victims of Alzheimer's disease.

Alzheimer Disease↗

PubMed2006

Interferon gamma quantification in cerebrospinal fluid compared with PCR for the diagnosis of tuberculous meningitis.

PURPOSE: To assess the utility of interferon gamma (INF-gamma) levels in cerebrospinal fluid (CSF), for the diagnosis of tuberculous meningitis (TBM), and compare these results with aPCR technique. METHODS: We studied CSF samples from patients with proven or probable TBM and a control group, composed by patients with other causes of meningitis and without meningitis. INFgamma levels were measured by radioimmunoassay. A PCR technique was performed using IS6110 primers. RESULTS: Of the 127 patients studied, 20 (15.6%) had TBM, 59 (46%) had meningitis of another aetiology and 49 (38.4%) had were HIV and non-HIV patients with normal CSF. The area below the ROC curve for interferon gamma levels in the diagnosis of TBM was 0.94. A cut-off of 6.4 IU/mL yielded a sensitivity of 70% and a specificity of 94%. False positive results were observed in 7 of the 59 patients (11.8%) with non-TB meningitis, (patients with herpetic meningoencephalitis and meningitis due to intracellular microorganisms). INF-gamma sensitivity was higher than PCR (70% vs. 65%). Both tests performed together showed higher sensitivity (80%) and specificity (92.6%). CONCLUSION: CSF INF-gamma levels (> 6.4 IU/mL) are very valuable in TBM diagnosis. PCR and INF-gamma could be simultaneously used to increase the diagnostic yield.

Adolescent↗

PubMed2002

High-resolution capillary zone and gel electrophoresis of structurally similar amphipathic glutathione conjugates based on interaction with beta-cyclodextrins.

The tripeptide glutathione is a prominent intracellular constituent that provides protection against genotoxic and carcinogenic electrophiles and is also a component of several biological signal substances. Glutathione conjugates, free glutathione, and glutathione disulfide contain charged amino acid residues, which contribute to solubility in aqueous media. However, the amphipathic nature of glutathione conjugates and the small differences that may distinguish the S substituents, pose analytical problems in their resolution. The present study demonstrates how homologous S-alkyl and S-benzyl conjugates of high structural similarity can be efficiently resolved by capillary electrophoresis. Inclusion of beta-cyclodextrins in the buffer or in a polyacrylamide gel affords baseline separation of the analytes. The separation methods described are applicable to enzyme assays in vitro and to the identification and quantification of glutathione conjugates of importance in toxicology and physiology. The contribution of beta-cyclodextrin to the separation is primarily based on interactions between its hydrophobic cavity and the S-alkyl and S-benzyl groups of the analytes.

Alkylation↗

PubMed1988

Resolution of complex fluorescence spectra recorded on single unpigmented living cells using a computerised method.

The identification and quantification of fluorescent compounds in a complex fluorescence spectra are always difficult, especially in the case of low signal:noise ratio. We propose a computerised method that allows the resolution of low light level complex fluorescence spectra into its components. Based on a linear combination of N possible characteristic fluorescence spectra, and using N weighting functions, this method allows the integration of fluorescence intensities over the entire fluorescence spectra and the generation of n equations with N unknowns. The compounds that participate in complex fluorescence spectra are identified and quantified. Because fluorescence intensities can be integrated we can resolve complex fluorescence spectra presenting a low signal:noise ratio. The reliability and sensitivity of our method are shown through examples of resolution of complex intracellular fluorescence of single living cells pretreated with benzo(a)pyrene. Depending on the cell type and treatment, two, four, or five components can be identified in the complex fluorescence spectra.

Animals↗

PubMed2026

Transcriptomic analysis reveals the molecular mechanisms underlying the inhibition of Mytilus edulis attachment by biofouling control agents.

This study combined acute toxicity assays, phenotypic quantification, and transcriptomic profiling to systematically investigate the inhibitory effects and molecular regulatory mechanisms of a novel alkylamine-based antifouling agent on survival, byssus secretion, and attachment behavior of juvenile Mytilus edulis. The 96 h-LC50 of the agent to juvenile M. edulis was 8.84 mg/L, and 10 mg/L of the agent completely inhibited mussel attachment within 24 h, significantly reducing byssal thread number, length, and diameter while increasing detachment frequency, resulting in irreversible attachment failure. Transcriptomic analysis identified 2746 differentially expressed genes, which were mainly enriched in pathways including signal transduction, immune defense, stress response, cytoskeleton organization, and protein binding. KEGG and GSEA enrichment revealed that the antifouling agent activated the MAPK stress signaling pathway, disturbed transcriptional regulation, and impaired intracellular homeostasis and cytoskeletal stability, thereby synergistically suppressing the expression of key byssal protein genes including mfp-1 and mfp-3 and ultimately blocking byssus synthesis and adhesion. This study clarifies the multi-pathway molecular mechanism underlying antifouling agent-induced attachment inhibition in M. edulis, and provides core molecular targets and theoretical support for developing efficient, specific antifouling activity, and potentially applicable marine antifouling technologies.

Animals↗