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Results for “Intracellular quantification”

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PubMed1995

Thyrotropin-releasing hormone gene expression in cultured anterior pituitary cells: role of gender.

The present studies were undertaken to investigate the effect of gender on thyrotropin-releasing hormone (TRH) gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male, female, or female pups that had been neonatally treated with testosterone propionate (TP), were cultured for up to 18 days in a modified DMEM/L-15 medium containing 10% fetal calf serum. TRH and AP hormones including GH, prolactin (PRL), luteinizing hormone (LH) and thyrotropin (TSH) were measured by RIA, proTRH mRNA was determined by in situ hybridization using a full-length riboprobe followed by quantification with a computer-assisted image analysis system. Cultures derived from female rats contained significantly (p < 0.01) higher amounts of TRH and secreted approximately twice (p < 0.01) as much TRH under basal conditions and in response to activators of the protein kinase A and C pathways, respectively. In situ hybridization studies revealed that 'female' cultures contained significantly higher amounts of proTRH mRNA compared to 'male' cultures. Computer-assisted image analysis demonstrated that proTRH mRNA levels were 3.5 times higher in 'female' compared to 'male' cultures (p < 0.01), an effect that was the result of a significantly higher number (3 times; p < 0.01) of cells expressing proTRH mRNA in 'female' cultures. Neonatal TP treatment did not affect either proTRH mRNA or TRH peptide levels. In vitro testosterone treatment resulted in a moderate rise (p < 0.05) of intracellular TRH accumulation in cultures from both sexes, however, proTRH mRNA levels remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1997

Quantification of ion transport in perfused rat heart: 133Cs+ as an NMR active K+ analog.

Proper ion balance between intra- and extracellular compartments is necessary for normal physiological function. Conversely, alterations in membrane ion transport occur in numerous pathological states. As a noninvasive, nondestructive spectroscopic technique, nuclear magnetic resonance (NMR) offers a powerful approach to the study of ion balance in intact biological systems. Unfortunately, rare NMR active nuclides that are isotopes of the 100% naturally abundant 23Na+ and 39K+ are not available for tracer kinetic studies of Na1 and K+ transport. However, Cs is a biologically active analog of K+, and the 100% naturally abundant NMR active 133Cs+ nuclide can be employed to examine K+ transport (Davis, D. G., E. Murphy, and R. E. London. Biochemistry 27: 3547-3551, 1988). The distinguishing feature of 133Cs+ is that it naturally gives two separate well-resolved NMR resonances for intra- and extra-cellular 133Cs+, permitting study of the time course changes of either of these compartments independent of the other. In this report, the experimental procedures and compartmental modeling formalism are developed that allow quantitative analysis of Cs+ membrane transport in the perfused rat heart. Intracellular 133Cs+ is shown to be 100% visible by solution-state NMR methods and its influx transport to be markedly inhibited by ouabain, a confirmation of findings previously reported by others. Intracellular 133Cs+ spin-lattice and spin-spin relaxation times at 7 T were determined to be 2.1 +/- 0.3 (SD)s (n = 8) and 0.065 +/- 0.007 (SD) s (n = 8), respectively, for T1 and T2. The rate constant for Na(+)-K(+)-ATPase pump dominated intracellular influx was measured to be 0.25 +/- 0.07 (SD) min-1 (n = 27) and that for efflux 0.005 +/- 0.001 (SD) min-1 (n = 14). The rate constant for 133Cs+ equilibration in the extracellular space at supraphysiological perfusate flow rate (20 ml/min) was found to be 4.6 +/- 0.9 (SD) min-1 (n = 20). Thus extracellular diffusion limitations do not dominate the 133Cs+ transport measurements.

Animals↗

PubMed2005

Differential expression of small-conductance Ca2+-activated K+ channels SK1, SK2, and SK3 in mouse atrial and ventricular myocytes.

Small-conductance Ca2+-activated K+ channels (SK channels, KCa channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca2+ with membrane potential. We recently reported for the first time the functional existence of SK2 (KCa2.2) channels in human and mouse cardiac myocytes. Here, we report cloning of SK1 (KCa2.1) and SK3 (KCa2.3) channels from mouse atria and ventricles using RT-PCR. Full-length transcripts and their variants were detected for both SK1 and SK3 channels. Variants of mouse SK1 channel (mSK1) differ mainly in the COOH-terminal structure, affecting a portion of the sixth transmembrane segment (S6) and the calmodulin binding domain (CaMBD). Mouse SK3 channel (mSK3) differs not only in the number of polyglutamine repeats in the NH2 terminus but also in the intervening sequences between the polyglutamine repeats. Full-length cardiac mSK1 and mSK3 show 99 and 91% nucleotide identity with those of mouse colon SK1 and SK3, respectively. Quantification of SK1, SK2, and SK3 transcripts between atria and ventricles was performed using real-time quantitative RT-PCR from single, isolated cardiomyocytes. SK1 transcript was found to be more abundant in atria compared with ventricles, similar to the previously reported finding for SK2 channel. In contrast, SK3 showed similar levels of expression in atria and ventricles. Together, our data are the first to indicate the presence of the three different isoforms of SK channels in heart and the differential expression of SK1 and SK2 in mouse atria and ventricles. Because of the marked differential expression of SK channel isoforms in heart, specific ligands for Ca2+-activated K+ currents may offer a unique therapeutic opportunity to modify atrial cells without interfering with ventricular myocytes.

Amino Acid Sequence↗

PubMed2005

Attenuation of hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats by aqueous extract of seed of Tamarindus indica.

Streptozotocin (STZ)-induced diabetic rats were divided into mild diabetic (MD) and severe diabetic (SD) on the basis of fasting blood glucose (FBG) levels. Diabetes was confirmed here by intravenous glucose tolerance test (GTT), biochemical assay of glycogen content in liver and skeletal muscle, glucose-6-phosphatase activity in liver, and serum insulin levels. Hyperlipidemia developed in these experimental diabetic rats was assessed by quantification of total cholesterol (TC), high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc) and triglyceride (TG) in serum. Aqueous extract of seed of Tamarindus indica was given to MD and SD rats at the dose of 80 mg and 120 mg/0.5 ml distilled water/100 g body weight/d respectively for 14 d. Significant attenuation of hyperglycemia was indicated by measuring FBG, glycogen level and glucose-6-phosphatase activity along with monitoring of intravenous GTT and serum insulin level. Similarly, correction of hyperlipidemia in diabetic rats after this extract supplementation was confirmed by significant reduction in the levels of above-mentioned hyperlipidemic indicators. Intravenous GTT was performed that highlights the antidiabetic action of this extract is not due to its effect on the intestinal rate of glucose absorption but may be due to modulation of intracellular glucose utilization in target organs. This study focus the efficacy of this extract for the management of experimental diabetes in rat model which may shed some light on the scientific basis of ancient herbal therapy in this line using this seed.

Animals↗

PubMed1997

Heat shock protein 70 expression in native and heterotopically transplanted rat hearts.

Heat shock proteins (hsp) are intracellular proteins that are rapidly synthesized in response to a variety of stress factors. Recent studies in rats have shown that these proteins can elicit a lymphocyte response during cardiac allograft rejection. We studied the expression of the inducible (i) and constitutive (c) forms of hsp70 in rat cardiac allograft and isograft recipients to evaluate their utility as indicators of transplant rejection. Heterotopic transplantation of rat hearts was performed, using Lewis to Lewis isografts and ACI to Lewis allografts. Sham-operated rats were used as controls. Transplanted isograft, allograft, and native hearts of the transplant recipients and their livers and spleens were harvested at 5 days posttransplant and analyzed for hsp70 (i) and (c) expression by Western blots. Seven animals were studied in each group. Isografts at 3 and 60 days and allografts at 8 days were also studied. Quantification of band densities was carried out by laser densitometry. Physiological function of the native hearts of the transplant recipients was studied using Langendorff preparations. High levels of hsp70 (i) were noted in the transplanted and native hearts of the transplant recipients but not in their livers or spleens or in the hearts of the sham-operated control animals. Myocardial function of the native hearts of the transplant recipients was not significantly different from that of the controls. Significantly higher levels of hsp70 (c) were present in mild and severely rejecting allografts compared with controls and nonrejecting isografts. In the rat model of heterotopic cardiac transplantation, high levels of hsp70 (i) in the native hearts of the allograft and isograft recipients suggest a transplant-related, cardiac-specific stress process, not previously described. Heat shock protein 70 (c) expression is significantly increased during early and late allograft rejection and may serve as an indicator of transplant rejection.

Animals↗

PubMed1989

Cerebral ischaemia studied by nuclear magnetic resonance spectroscopy.

In this review, we assess the role of nuclear magnetic resonance (NMR) spectroscopy as a noninvasive method of studying metabolism in cerebral ischaemia. Phosphorus-31 NMR provides a monitor of intracellular pH and energy metabolites, including ATP, phosphocreatine, and inorganic phosphate, while other nuclei, including 1H, 13C, 19F, and 23Na can give additional information about several aspects of brain metabolism and physiology. For example, 1H NMR not only provides excellent images, but may also be used to monitor a range of metabolites, including lactate and several amino acids. Comparisons are made with the large body of information that is available from more traditional methods of studying metabolism. Emphasis is placed on the correlation of NMR data with parallel measurements of regional blood flow, tissue oxygenation, oedema, electrical activity, and tissue damage. Technical aspects of NMR are discussed where appropriate; for example, in relation to the range of metabolites that are accessible to study, the spatial resolution that is available for studies of focal lesions, problems arising from tissue heterogeneity, and quantification of metabolite levels. Applications in animal models and in humans are discussed; these primarily involve the 31P nucleus, but for the future it appears that 1H NMR studies offer particular promise.

Brain Ischemia↗

PubMed1986

P NMR evaluation of hypoxic stress in brain of animal models.

Quantification by NMR of hypoxic stress is best afforded by the ratio of phosphocreatine (PCr) to inorganic phosphate (Pi). The thin cranium and sternum of neonates affords ideal conditions for NMR evaluation of impaired oxidative metabolism. Brains of animal models (dog and cat) are best studied with muscles retracted with the rf coil placed on the bare skull. 31P and 1H (lactate) signals are time shared with a doubly tuned coil. Servo-stabilized hypoxia gives simplified steady state analysis. Hypoxic insults of the brain are evaluated by the integral of the deviation of the PCr/Pi ratio from the normoxic values. Generally, a deviation of 1 unit of PCr/Pi for an interval of 3 hr will lead to a severe metabolic damage and recovery from hypoxia is often 30-fold delayed. Comprehensive monitoring of energy metabolism (PCr/Pi), respiratory chain redox states, intracellular pH and lactate accumulation allow the calculation of integrated pH and lactate "insults" and thus offer new insights on the effects of hypoxia on brain oxidative metabolism.

Animals↗

PubMed1986

Aging in the AXC/SSh rat: characterization of moderately abundant ventral prostate proteins showing age-dependent diminution and one protein exhibiting age-invariant content.

To determine whether prior demonstrations of age-related decrements in prostate content of minor, androgen regulated proteins represent a generalized phenomenon, we validated a denaturing polyacrylamide gel electrophoretic protocol for separation and quantification of moderately abundant ventral prostate cytoplasmic proteins. We established age-related, progressive 3- to 3.5-fold decreases in prostate content of proteins of 90, 79, 63, and 58 kDa and found that content of a 46 kDa protein was age-invariant. The amount of 90 and 46 kDa proteins was not significantly altered, whereas the level of 79, 63 and 58 kDa proteins decreased during 72 h post-orchiectomy of 3-month-old rats. Testosterone injection of intact 26-month-old rats caused an average 2-fold increase in 90, 79, 63, and 58 kDa protein content and did not affect 46 kDa protein level. Because we demonstrated the 46 kDa protein is not a secretory protein, absence of an affect of aging or testosterone on prostate content is not due to secretion mediated inaccessibility to intracellular processing. The apparent relation between age and prostate content of these proteins is not a consequence of potential age-related changes in ventral prostate cell content or distribution because biochemical and histologic analyses show this does not significantly occur. Our studies establish age-related decreases in ventral prostate content of moderately abundant, androgen responsive proteins and show that content of at least one protein is age- and androgen-independent. It remains to be determined whether these findings reflect direct effects of gene regulation.

Aging↗

PubMed2001

Structure-function investigation of the interaction of 1- and 2-substituted 3-hydroxypyridin-4-ones with 5-lipoxygenase and ribonucleotide reductase.

The structural and physiochemical properties of 3-hydroxypyridin-4-one chelators (HPOs) which influence inhibition of the iron-containing metalloenzymes ribonucleotide reductase (RR) and 5-lipoxygenase (5-LO) have been investigated. HPOs with substituents at the 1- and 2-positions of the pyridinone ring have been synthesized, and their inhibitory properties compared with those of desferrioxamine (DFO). Varying the alkyl substituents does not affect the affinity constant of these ligands for iron(III), but permits a systematic investigation of the effect of hydrophobicity and molecular shape on inhibitory properties. The inhibition of RR was monitored, indirectly by measuring tritiated thymidine incorporation into DNA and directly by the quantification of the EPR signal of the enzyme tyrosyl radical. 5-LO inhibition was examined spectrophotometrically, measuring the rate of linoleic hydroperoxide formation by soybean lipoxygenase. The results indicate that the substituent size introduced at the 2-position of the HPO ring is critical for determining inhibition of both enzymes. Large substituents on the 2-position, introduce a steric factor which interferes with accessibility to the iron centers. These studies have identified chelators such as 1,6-dimethyl-2-(N-4',N-propylsuccinamido)methyl-3-hydroxypyridin-4-one (CP358), which causes only a 10% inhibition of 5-LO after 24 h of incubation at 110 microm IBE (iron-binding equivalents) in comparison to simple dialkyl HPOs such as Deferiprone (CP20) which cause up to 70% inhibition. Using EPR spectroscopy, CP358 inhibits RR at a slower rate than CP20, while chelating intracellular iron(III) at a similar rate, a finding consistent with an indirect inhibition of the tyrosyl radical. However, hepatocellular iron is mobilized at a faster rate by CP358 (P < 0.001). These findings demonstrate that it is possible to design bidentate HPOs which access intracellular iron pools rapidly while inhibiting non-heme iron-containing enzymes relatively slowly, at rates comparable to DFO. It is anticipated that such compounds will possess a superior therapeutic safety margin to currently available bidentate HPOs.

Arachidonate 5-Lipoxygenase↗

PubMed1999

Quantification of glucose transport and phosphorylation in human skeletal muscle using FDG PET.

UNLABELLED: PET with 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) is used for quantifying glucose metabolism in brain and myocardium in vivo. We developed and validated a similar procedure for the quantification of the two initial steps of glucose metabolism in skeletal muscle in vivo. METHODS: The measurement protocol was first optimized by computer simulations. In addition to the accuracy in sampling plasma input and tissue time-activity curves, precise determination of the fractional blood volume, that is, the extracellular tissue volume fraction, plays a key role in correctness of the determined model constants. The optimized protocol was subsequently used to estimate transmembrane muscular glucose transport and hexokinase activity in six human subjects with normal or altered glucose utilization. PET was performed during the steady state of an euglycemic hyperinsulinemic clamp. RESULTS: A three-compartment model provides a better description of the experimental data than a two- or four-compartment model. Glucose clearance from the extracellular compartment into the skeletal muscle cell (K1) ranges from 0.024 to 0.093 mL/g/min. The intracellular glucose phosphorylation rate (k3) varies between 0.030 and 0.142 min(-1). The regional muscular glucose utilization, as calculated from the determined model parameters, lies between 10.7 and 83.3 micromol/kg/min and correlates with the whole-body glucose utilization as independently determined (R2 = 0.83; P < or = 0.01). CONCLUSION: We demonstrate by computer simulations that a three-compartment model can be used to characterize the first two steps of glucose metabolism in skeletal muscle. An optimized measurement protocol is developed and applied to experimental data. This experimental approach should be appropriate to test whether glucose transport or hexokinase activity is altered in disorders of muscular glucose utilization.

Adult↗

PubMed2006

Quantification of VGF- and pro-SAAS-derived peptides in endocrine tissues and the brain, and their regulation by diet and cold stress.

Two novel granin-like polypeptides, VGF and pro-SAAS, which are stored in and released from secretory vesicles and are expressed widely in nervous, endocrine, and neuroendocrine tissues, play roles in the regulation of body weight, feeding, and energy expenditure. Both VGF and pro-SAAS are cleaved into peptide fragments, several of which are biologically active. We utilized a highly sensitive and specific radioimmunoassay (RIA) to immunoreactive, pro-SAAS-derived PEN peptides, developed another against immunoreactive, VGF-derived AQEE30 peptides, and quantified these peptides in various mouse tissues and brain regions. Immunoreactive AQEE30 was most abundant in the pituitary, while brain levels were highest in hypothalamus, striatum, and frontal cortex. Immunoreactive PEN levels were highest in the pancreas and spinal cord, and in brain, PEN was most abundant in striatum, hippocampus, pons and medulla, and cortex. Since both peptides were expressed in hypothalamus, a region of the brain that controls feeding and energy expenditure, double label immunofluorescence studies were employed. These demonstrated that 42% of hypothalamic arcuate neurons coexpress VGF and SAAS peptides, and that the intracellular distributions of these peptides in arcuate neurons differed. By RIA, cold stress increased immunoreactive AQEE30 and PEN peptide levels in female but not male hypothalamus, while a high fat diet increased AQEE30 and PEN peptide levels in female but not male hippocampus. VGF and SAAS-derived peptides are therefore widely expressed in endocrine, neuroendocrine, and neural tissues, can be accurately quantified by RIA, and are differentially regulated in the brain by diet and cold stress.

Animals↗

PubMed2004

Determination of cellular nicotinic acid-adenine dinucleotide phosphate (NAADP) levels.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725-729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor-ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.

Animals↗

PubMed2000

Isoform expression of the sarcoplasmic reticulum Ca2+ release channel (ryanodine channel) in human myocardium.

The Ca2+ release channel of the sarcoplasmic reticulum (SR) is essential for the release of Ca2+ from intracellular stores and is expressed widely in various excitable cells. It plays a key role particularly in excitation contraction coupling in myocytes in skeletal and cardiac muscle. Three isoforms of the SR Ca2+ release channel have been cloned. Recently coexpression of different isoforms was reported in different animal species and various tissues. In human cardiac tissue, however, isoform expression is not yet established. Therefore the aim of this study was to characterize isoform expression of the SR Ca2+ release channel in the human heart. We examined specific isoform expression of mRNA and proteins of the SR Ca2+ release channel in the four different chambers of the heart and the interventricular septum from explanted human hearts from nonfailing organ donors (n=8). Reverse transcriptase PCR from total cardiac RNA with isoform specific primers and western blots from myocardial homogenates with isoform specific antibodies were performed. Quantification of protein expression was achieved by densitometric scanning and computer analysis and is expressed as densitometric units per microgram of protein. A single band DNA signal was detected by reverse transcriptase PCR for the skeletal isoform 1 and the cardiac isoform 2 and isoform 3 in all regions of the human heart investigated. Specific protein expression was detected in all five myocardial regions of the human heart in western blots for the skeletal isoform I and cardiac isoform 2, and a weaker specific band was also detectable for isoform 3 of the SR Ca2+ release channel. Quantification of protein expression showed significant (P=0.008) lower expression of isoform 1 in the right ventricle (42+/-4 densitometric units/g tissue) and similar expression in all other regions (right atrium 58+/-3; septum 51+/-5, left atrium 54+/-5; left ventricle 51+/-6). Isoform 2 of the SR Ca2+ release channel was also significantly lower (P=0.001) in the right ventricle (33+/-4 densitometric/g tissue) and similar in the other heart chambers (right atrium 42+/-5: septum 41+/-3, left atrium 52+/-6, left ventricle 42+/-3). Differences in isoform 3 of the SR Ca2+ release channel for the various myocardial regions did not reach significant levels (right atrium 45+/-6, right ventricle 38+/-5, septum 49+/-8, left atrium 46+/-7, and in left ventricle 45+/-3 densitometric units/g tissue). In conclusion, all three isoforms of the SR Ca2+ release channel were determined in the human heart at both mRNA and protein levels with different quantitative expression in the different heart chambers. Coexpression of the three different isoforms with different functional properties might increase the complexity of regulation of excitation contraction coupling in the human heart in a chamber specific mode.

Adult↗

PubMed2003

Fabry disease: diagnosis and treatment.

Fabry disease is an X-linked lysosomal storage disorder that results from a deficiency of the enzyme alpha-galactosidase A (alpha-Gal A). The lack of alpha-Gal A causes an intracellular accumulation of glycosphingolipids, mainly globotriaosyceramide (GL3). Affected organs include, among others, the vascular endothelium, heart, brain, and kidneys, leading to end-stage renal disease (ESRD). Since Fabry disease cannot be cured at present, clinical management is symptomatic. Enzyme replacement therapy (ERT) with recombinant alpha-Gal A has been introduced as a new therapeutic option for the treatment of Fabry patients. Short-term (one year) clinical studies have positively correlated ERT with improvement of clinical symptoms and microvascular endothelial cell clearance. Treatment outcome concerning severe organ manifestations such as proteinuria and renal function impairment, left ventricular hypertrophy, and heart failure in the long run has yet to be shown. In our studies we used sensitive and noninvasive techniques such as ultrasound-based strain rate imaging and magnetic resonance imaging (MRI), combined with MR-spectroscopy (MR-S), for the quantification of functional abnormalities at an early stage of the disease and during long-term follow-up. Future issues should determine the appropriate timing to start therapy and how children and heterozygous females should be managed. Given the diagnostic and therapeutic potential today, it is of importance to identify patients at an early stage and to start therapeutic intervention before progression of organ damage is inevitable.

Fabry Disease↗

PubMed2006

Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.

The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD.

Animals↗

PubMed2005

The diverse signaling network of EGFR, HER2, HER3 and HER4 tyrosine kinase receptors and the consequences for therapeutic approaches.

The HER family of receptor tyrosine kinase couples binding of extracellular growth factor ligands to intracellular signal transduction pathways, contributing in this fashion to the ability of the cell to respond correctly to its environment. The HER family and its ligands are critically involved in the carcinogenesis of the mammary gland. Abnormal function of the members of HER family resulting in receptor hyper-activation (due to gene amplification, protein overexpression or abnormal transcriptional regulation) has been linked with breast cancer prognosis. It is also extensively studied as the predictive factor and target for therapy. There are clinical indications supporting the concept that none of the receptors: EGFR, HER2, HER3 and HER4 can be considered as the stand-alone receptor in breast cancer development and clinical course of the disease. There is a growing body of evidence that cooperation between them contributes to more aggressive tumor phenotype and influences the response to therapy. This underlines the importance of quantification of all HER family members and indicates the urgent need for implementation of methods that can efficiently and reliably examine four HER receptors as a whole panel in breast cancer patients.

Animals↗

PubMed1991

In vivo sodium-23 MRI in brain tumors: evaluation of preliminary clinical experience.

In vivo sodium-23 magnetic resonance (MR) imaging in the head was performed in ten normal volunteers and six patients with brain tumors on a commercially available 1.5 Tesla whole-body MR system. Although sodium MR signal from the brain parenchyma could hardly be detected on routine spin echo (SE) pulse sequence, free induction decay (FID) image with echo time of 1.9 msec demonstrated MR signal in the normal brain tissue. On the other hand, extracellular sodium of cerebrospinal fluid, large venous sinus, and vitreous humor offered high sodium MR signal intensity due to the quantity of sodium and the relatively longer transverse relaxation time (50-60 msec) compared to that of intracellular sodium (less than a few msec). Strong signal was obtained in gliomas with superior contrast, though the delineation of the tumor tissue from edema was as difficult as when seen on proton T2 weighted SE image. Meningioma itself gave much less of a sodium MR signal compared to glioma. Peritumoral edema associated with meningioma showed a markedly high sodium MR signal which was more prominent adjacent to the tumor, although proton SE image revealed the peritumoral edema as homogeneous. Pathological tissues such as brain tumors and edema could be readily depicted as significantly high sodium-23 MR signal, apparently different from normal brain tissues. The current status of in vivo sodium MR imaging is discussed. The authors concluded that early clinical experience of in vivo sodium-23 MR imaging brought promising results in the evaluation of brain tumors and edema; otherwise, discrimination and quantification of multiple T2 components and the improvement of spatial resolution are desperately needed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1998

Lectin-mediated drug targeting: quantification of binding and internalization of Wheat germ agglutinin and Solanum tuberosum lectin using Caco-2 and HT-29 cells.

For the potential use of Wheat germ agglutinin (WGA) and Solanum tuberosum lectin (STL) as auxiliary excipients for targeting drugs to colonocytes, the number of Caco-2 and HT-29-bound lectins was determined by fluorimetry using fluorescein-labelled derivatives of the N-acetylglucosamine-specific lectins. After 1 h of incubation, the WGA-binding capacity of 5 x 10(4) Caco-2 cells was 26.9 +/- 0.5 pmol at 4 degrees C and 27.2 +/- 1.0 pmol at 37 degrees C respectively. In comparison, 19.5 +/- 2.9 pmol (37 degrees C) and 16.7 +/- 0.9 pmol (4 degrees C) WGA were bound within 1 h to 5 x 10(4) HT-29 cells referring to about 80% of the total amount of WGA bound within 4 h of incubation. In contrast, binding of STL to the colon carcinoma cell lines was independent of incubation times and temperatures tested exhibiting a binding rate of 8.4 +/- 0.6 pmol (HT-29) and 9.9 +/- 0.8 pmol (Caco-2) STL/5 x 10(4) cells. As determined by flow cytometry, non-specific binding is lower than 1.0% (WGA) and 3.4% (STL). Uptake and intracellular accumulation of the lectins were investigated by confocal laser scanning microscopy at 4 degrees C and 37 degrees C respectively. A decrease of initially membrane-bound lectins concurrent with increasing cytoplasmic enrichment by time was observed by digital cell image analysis. Due to specific and numerically sufficient adhesion as well as internalization, WGA and STL are anticipated as targeting tools in lectin-mediated drug delivery systems.

Acetylglucosamine↗