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Biomedical subjects

Y Shechter

Publications and source records attributed to Y Shechter.

At least 109 records · Page 6Linked to original sources
PubMed1981

Is routine crossmatching for two units of blood necessary in elective surgery?

The usual preparation for many elective procedures calls for typing and crossmatching for 2 units of blood. Most of the blood prepared is not used. Every unit of blood kept for a specific patient is removed from the blood bank pool, thereby reducing its resources. Fourteen hundred of the most frequent elective operations were examined with regard to their blood utilization. A transfusion index was developed to determine whether preoperative crossmatching was justified. Applying the index to 1,400 elective procedures, it was found that only vascular procedures justified preoperative crossmatching. The safety of transfusion (elective and emergency) based on type and antibody screening is explained. The constant increase in blood consumption (as for open heart surgery) and the small increase in blood donations has created a blood shortage. It is suggested that surgeons stop requesting crossmatching for 2 units for those elective procedures in which blood is rarely used. This would decrease the workload in the blood bank and increase the blood bank pool without compromising patients' safety.

Blood Grouping and Crossmatching↗
PubMed1980

Mobility, clustering, and transport of nerve growth factor in embryonal sensory cells and in a sympathetic neuronal cell line.

We have prepared a fluorescent conjugate of nerve growth factor (NGF) containing 8--10 rhodamine molecules attached to free carboxyl groups of the protein. This analogue retained full binding capacity toward NGF receptors, full antigenic properties, and the potency to stimulate the differentiation of embryonal chicken sensory ganglia cells in vitro. We have used this analogue to study the mobility and distribution of NGF receptors on embryonal chicken sensory cells from dorsal root ganglia and on a pheochromocytoma cell line (PC-12) that responds to NGF by differentiating along a neuronal pathway. The rhodamine conjugate of nerve growth factor (R-NGF) binds initially to diffusely distributed mobile receptors (D approximately 8 X 10(-10) cm2/sec) on immature sensory and PC-12 cells. At 37 degrees C, the NGF receptor complexes cluster and form immobile visible patches. These patches undergo endocytosis in a process that consumes metabolic energy. Methylamine blocks the formation of visible patches of NGF and the receptors remain dispersed and mobile at 37 degrees C. On differentiated chicken sensory cells, R-NGF binds to diffusely distributed mobile receptors and to aggregated immobile binding sites. These clusters are localized at the tip of the axon, along the axon, and in the main body. The NGF molecules that are internalized at the tip of the axon are transported retrogradely from the peripherey to the cell body.

Animals↗
PubMed1979

13C NMR analysis of methionine sulfoxide in protein.

The 13C epsilon NMR signal of methionine sulfoxide is 22.6 ppm downfield from that of methionine. This affords a method by which the extent of methionine oxidation can be determined in intact protein. We demonstrate the utility of this approach with beta-galactosidase enriched with 13C in its methionine methyls.

Chloramines↗
PubMed1979

Insulin receptor: covalent labeling and identification of subunits.

Two methods were used to label insulin receptors covalently with 125I. In the first, an aryl azide derivative of insulin, 125I-labeled 4-azido-2-nitrophenyl-insulin, was synthesized and used to photolabel the binding region of the insulin receptor in rat liver membranes and human placenta membranes. In the second, insulin receptors were purified from rat liver membranes and labeled with 125I by use of chloramine-T; this method presumably has no specificity for the binding region of the receptor. The proteins labeled by both methods were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after or without reduction by dithiothreitol. The photoaffinity label specifically labeled a single band in both liver and placenta that had an apparent molecular weight of 135,000 after reduction. A band with similar mobility was present in the chloramine-T-labeled preparation, which also contained a second major band with an apparent molecular weight of 45,000. Without reduction, both methods resulted in a single labeled band with an apparent molecular weight of about 310,000. These results indicate that the insulin receptor of both liver and placenta has a subunit of molecular weight 135,000 that binds insulin and that the receptor may be composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds.

Affinity Labels↗
PubMed1979

Modulation of binding and bioactivity of insulin by anti-insulin antibody: relation to possible role of receptor self-aggregation in hormone action.

Incubation of physiological concentrations of 125I-labeled insulin with liver membranes in the presence of anti-insulin IgG results in a 7- to 15-fold increase in the specific binding of the hormone. The low-affinity/high-capacity binding sites are replaced by an apparently homogeneous class of high-affinity sites, and the nonlinear Scatchard plots are converted to linear plots without a change in the maximum number of binding sites. Similarly, the binding of insulin to receptors in 3T3 fibroblasts is increased substantially in the presence of anti-insulin antibody, and the biological activity of subactive concentrations of insulin is enhanced by antibody in these cells. However, the affinity of 125I-labeled epidermal growth factors (EGF) in fibroblasts is not affected by anti-EGF IgG. In adipocytes anti-insulin IgG in the same concentration range only inhibits the binding of insulin and suppresses insulin-mediated glucose oxidation. Monovalent Fab' fragments from anti-insulin IgG inhibit the binding of the hormone, indicating that the enhancement of binding in liver membranes and fibroblasts requires the bivalency of the antibody.

Animals↗
PubMed1978

Enhancement of alpha-chymotrypsin-catalyzed hydrolysis of specific p-nitroanilide substrates by 4-phenylbutylamine derivative of hen egg-white lysozyme.

Modification of hen egg-white lysozyme by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in presence of 4-phenylbutylamine yielded derivatives, which contained 0.6--0.7 modified residues and retained about 60% of the original activity. Kinetic studies revealed that the modified-lysozyme increases approx. 20-fold the kcat of hydrolysis of SucGly2Phe-4-nitroanilide by alphachymotrypsin, without changing the Km. The apparent dissociation constant of phenylbutylamine-modified lysozyme . chymotrypsin complex was found to be 0.03 mM and independent of substrate concentration. The accelerating effect of the modified lysozyme was also observed with other p-nitroanilide substrates of alpha-chymotrypsin. However, the hydrolysis of other substrates, acylation by active site titrant or inhibition by irreversible or competitive inhibitors were uneffected. The enhancing effect of the modified lysozyme seems to be very specific since other chymotrypsin-like enzymes, or serine proteinases except delta-chymotrypsin, were not influenced and phenylbutylamine derivatives of alpha-lactalbumin or ribonuclease were lacking any enhancing effect. Smaller, but significant enhancing effect was found also in lysozyme substituted by benzylamine, beta-phenylethylamine and tryptamine and in inactive derivatives of lysozyme substituted by phenylbutylamine. Competitive inhibitors of lysozyme such as N-acetyl-D-glucose amine oligomers, (GlcNAc)2 and (GlcNAc)3 abolished partially the accelerating effect of phenylbutylamine-modified lysozyme, indicating that the substituted group is located in the vicinity of the binding site.

Anilides↗
PubMed1978

Quantitation of methionyl peptides in nanomole quantities by a fluorometric method.

A quantitative and highly specific method to determine low concentrations of methionyl peptides, which do not contain tryptophan or cysteine residues, has been developed. The method is based on the stoichiometry and selectivity of N-chlorosuccinimide (NCS) towards methionine and N-acetyltryptophan. N-Chlorosuccinimide reacts with N-acetyltryptophan in a 1:1 ratio to produce the N-acetyl-2-oxindolealanine--a derivative essentially devoid of fluorescence. The decrease in fluorescence intensity is approximately linear with respect to the NCS concentration. Preincubation of NCS with methionine or methionyl peptide consumes a stoichiometric amount of the reagent and the unreacted NCS is quantitated by the decrease in fluorescence intensity resulting upon incubation of the mixture with 1 eq of N-acetyltryptophan. Less than 1 nmol of methionyl peptide can be accurately quantitated by this method.

Indicators and Reagents↗
PubMed1978

Collection of insulin, EGF and alpha2-macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization.

We have used video intensification microscopy to observe fluorescent derivatives of insulin, epidermal growth factor and alpha2-macroglobulin added to Swiss 3T3-4 cells. At 4 degrees C, each of these polypeptides binds diffusely to specific receptors on the cell surface. When the cells are warmed to 23 or 37 degrees C, the bound insulin epidermal growth factor or alpha2-macroglobulin rapidly forms patches on the cell surface and is internalized. Using fluorescein-labeled alpha2-macroglobulin and rhodamine-labeled derivatives of insulin and epidermal growth factor, we show that all three polypeptides are internalized within the same vesicles by a common pathway. The mechanism for the internalization of these molecules is discussed.

Cell Line↗
PubMed1978

Quantitative determination of the lateral diffusion coefficients of the hormone-receptor complexes of insulin and epidermal growth factor on the plasma membrane of cultured fibroblasts.

Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are mobile on the cell surface, with similar diffusion coefficients, D approximately (3--5) x 10(-10) cm2/sec at 23 degrees C. Increasing the temperature to 37 degrees C results in rapid receptor immobilization. The immobilization is attributed to aggregation of hormone-receptor complexes, their internalization, or a combination of both processes.

Azides↗
PubMed1978

Epidermal growth factor: biological activity requires persistent occupation of high-affinity cell surface receptors.

The enhancement of DNA synthesis by epidermal growth factor (EGF) in cultured human fibroblasts is demonstrable 24 hr after incubation of the cells at 37 degrees C with very low concentrations (0.83 nM) of the hormone for very short periods (30 min) followed by thorough washing of the cells to remove the free hormone in the medium. This effect must result from persistent, extraordinarily tight binding of the hormone to surface receptors, because the addition of specific, purified anti-EGF IgG as late as 8 hr after initial hormone exposure can completely reverse the biological effects of the hormone. This causes only a slight (but significant) increase in the rate of dissociation at 37 degrees C of the cell-bound (125)I-labeled EGF at low occupancy. Together with the fact that in the presence or absence of antibody virtually all of the demonstrable cell-bound (125)I-labeled EGF can be shown to dissociate from the cell during a period as short as 2-3 hr, the data suggest the possibility that the biological effects of this hormone may be mediated by occupation of only a negligible fraction of very high affinity binding sites. Thus, the processes of hormone internalization, degradation, and "down regulation" may be irrelevant to the effects of the hormone on DNA synthesis. For this effect the crucial and limiting processes appear to be strictly related to the continuous and persistent occupation of cell surface receptors.

Antigen-Antibody Reactions↗
PubMed1978

Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin.

Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.

Adipose Tissue↗
PubMed1978

Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells.

We have studied in detail the binding of fluorescent derivatives of insulin and epidermal growth factor to 3T3 fibroblasts. We have used two types of fluorescent analogues of insulin and epidermal growth factor: highly fluorescent derivatives which have seven to eight rhodamine molecules or fluorescent derivatives which have a single rhodamine molecule per one molecule of insulin or epidermal growth factor. Both types of analogue retained substantial binding affinity as determined by radioreceptor assays and biological activity. The cells labeled with the fluorescent analogues were visualized with a sensitive video intensification microscopic system that enabled us to directly observe the location of the fluorescent hormone on the surface and within the living fibroblasts. We found that both insulin and epidermal growth factor initially bound diffusely to the cell surface and, at 4 degrees , remained dispersed. Within a few minutes at 23 degrees or 37 degrees the hormone-receptor complexes aggregated into patches that could be readily removed by trypsin but not by excess native hormone. The hormone-receptor complexes, which were initially mobile in the plane of the membrane, become immobilized later as the consequence of the receptor aggregation or internalization. Within approximately 30 min at 37 degrees , much of the labeled hormone was found within the cell in endocytic vesicles that moved about in the cytoplasm in a saltatory manner. The aggregation and immobilization of the hormone-receptor complexes could be due to either hormone-hormone interactions on the cell membrane or a hormone-induced conformational change in the hormone-receptor complex. Aggregation and internalization of hormone-receptor complexes could be associated with certain aspects of hormone action, hormone degradation, down regulation of receptors, or negative cooperativity of hormone binding.

Cell Line↗
PubMed1978

Metabolic activity of developing rabbit lung.

The uptake and metabolism of [3H]leucine, [U-14C]glucose, and [3H]palmitate were studied in rabbits aged --3, 1, 7, 28, and 90 days up to 1--1.5 years. Although lung composition did not change markedly, there were great differences in lung metabolism between the perinatal period (late fetal and newborn) and later stages of development. Leucine incorporation into lung protein was highest (3.1--3.4 nmol/mg protein/hr) in late fetal and newborn rabbits and decreased rapidly thereafter. Palmitic acid incorporation decreased during the first week after birth from 150 nmol/100 mg/hr to 85 nmol/100 mg/hr at 7 days of age; it increased thereafter to 170 nmol/100 mg/hr at 4 weeks of age and remained at that level throughout the entire period studied. Glucose uptake and lactate production were higher in fetal lungs than in all other age groups. Lipid biosynthesis from glucose was 2--4 times higher in fetal lungs than at all other ages; furthermore, more than 60% of glucose carbon atoms channeled into lipid was incorporated into fatty acids, whereas at all other ages glucose was chiefly a precursor of lipid glycerol.

Animals↗
PubMed1977

Selective covalent binding of methionyl-containing peptides and proteins to water insoluble polymeric reagent and their regeneration.

A polymeric reagent of the type P approximately NHCOCH2Cl (where P is Bio-Gel P-100) was prepared. This polymer covalently bound peptides and proteins specifically at methionine residues under acidic conditions in the presence of a small amount of sodium iodide. Treatment of the polymer-peptide conjugate with 2-mercaptoethanol resulted in essentially complete removal of the peptide with regeneration of intact methionyl residues. In an alternative way, the polymer was suspended for 2 h in boiling water. This treatment resulted in the conversion of the bound methionyl residues to homoserine residues and cleavage and liberation of the bound peptides. The polymeric reagent was successfully applied to the separation of methionyl peptides from peptide mixtures and for specific covalent binding of enzymes and biologically active proteins via their exposed methionyl residues, with the retention of their biological activity.

Acrylamides↗