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Biomedical subjects

R L Klein

Publications and source records attributed to R L Klein.

At least 91 records · Page 5Linked to original sources
PubMed1984

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether.

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.

Animals↗
PubMed1984

Biobrane--a useful adjunct in the therapy of outpatient burns.

Fifty-five patients with partial thickness burns ranging from 1% to 10% of their body surfaces have been treated with Biobrane (Woodroof Laboratories, Santa Ana, CA) dressings on an outpatient basis. This material is a biosynthetic skin prosthesis that was introduced commercially in 1979. Advantages of Biobrane over other conventional dressings in outpatient burn care have been the significant pain relief, reduced number of dressing changes, decreased patient visits, and satisfactory epithelization in 7 to 14 days, ease of application and removal, and cost effectiveness. The dressing must be placed on clean wounds judged to be partial thickness in depth. Topical antibiotics are unnecessary in the care of these patients.

Burns↗
PubMed1983

Hydrophilic form of dopamine beta-hydroxylase from purified noradrenergic vesicles. Activities and comparisons.

Bovine splenic nerve and adrenal medulla were used as homologous sources of dopamine beta-hydroxylase permitting the isolation of enzyme specific to a purified fraction of large dense cored noradrenergic vesicles and chromaffin granules, respectively. The hydrophilic (water soluble) form of the enzyme was purified to homogeneity on the bases of gel electrophoresis, isoelectric focusing, and double immunodiffusion tests from the physical lysates of the vesicles and granules. Amino acid analyses suggest that the hydrophilic dopamine beta-hydroxylase is the predominant form in the nerve vesicles. It has higher neutral and lower hydrophobic amino acid group residues when compared to the adrenomedullary enzyme prepared in this and most other laboratories. Among the neutral amino acids, this difference appears to reflect approximately 40% higher serine and glycine contents, and among the hydrophobic amino acids it may reflect in part approximately 25% lower leucine content. Although the terms hydrophilic and amphiphilic can be properly applied to certain chemical properties of the D beta H forms, it is not at all certain that these terms can be used quantitatively to describe the matrix and membrane associated forms of the enzyme, respectively.

Amino Acids↗
PubMed1983

Local modulation of neurotransmitter release in bovine splenic vein.

Bovine splenic vein has an abundant sympathetic innervation. Isolated strips were used to examine whether autoinhibition of norepinephrine release from the noradrenergic nerve terminals could be demonstrated under various experimental conditions and whether additional local regulatory modulators of transmitter release could also be implicated. In particular, the possibility of a histamine interaction with presynaptic inhibitory receptors was examined because ultrastructural evidence disclosed a close spatial relationship between mast cells and noradrenergic nerve terminals in the vessel wall. To investigate the presence of presynaptic alpha-receptors the competitive blocking agent phentolamine was included in the superfusion medium at concentrations ranging from 1 to 50 microM during electrical field stimulation at frequencies between 1 and 10 Hz. Transmitter outflow was measured as fractional tritium release. Low frequency stimulation (1 Hz) with 1 microM phentolamine resulted in the typical increase in norepinephrine release characteristics for presynaptic alpha-receptor inhibition. In contrast, high frequency (10 Hz) stimulation in the presence of 50 microM phentolamine caused an unexpected decrease in norepinephrine outflow. This unusual result can be explained by additional pharmacological actions of phentolamine unrelated to alpha-receptor blockade, e.g. histamine release from the mast cells which subsequently can act on presynaptic inhibitory histamine receptors. This effect, manifested at higher phentolamine concentrations, would overcome the alpha-receptor blockade. The presence of histamine receptors was supported by the results from electrical stimulation in the presence of exogenous histamine. Histamine decreased norepinephrine outflow while increasing basal tension and the contractile response of the vein strip. Unexpectedly, these effects appeared to be mediated by histamine receptors of the H1-type because they were reduced after pyrilamine but unaffected by agonists and antagonists to receptors of the H2-type. It is speculated that interactions between mast cells and noradrenergic nerve terminals may serve to maintain homeostasis in the bovine splenic vein.

Animals↗
PubMed1983

Cholesterol absorption and transport in thoracic duct lymph lipoproteins of nonhuman primates. Effect of dietary cholesterol level.

The effect of dietary cholesterol level on cholesterol absorption and its subsequent transport in thoracic duct lymph lipoproteins was studied in two species of nonhuman primates, namely the African green monkey (Cercopithecus aethiops) and the cynomolgus macaque (Macaca fascicularis). Each animal served as its own control because each received sequential, intraduodenal infusions of two fat-rich liquid diets that differed only in the amounts of cholesterol. The percentage of dietary cholesterol absorbed was calculated by dividing the rate of appearance of exogenous cholesterol radioactivity in lymph by the rate of infusion of dietary cholesterol radioactivity at a time when lymph cholesterol specific activity was constant relative to that of diet. The percentage of dietary cholesterol absorbed was similar during both diet infusions in African green monkeys but was significantly decreased during the high cholesterol diet infusion in cynomolgus macaques. Rates of appearance of lymph total cholesterol mass were significantly increased during infusion of high cholesterol diets due to a statistically significant increase in cholesteryl ester transport rates. This increase was due in large part to the preferential esterification of exogenous cholesterol that was incorporated into lymph chylomicrons and VLDL. The rate of appearance in lymph of exogenous cholesterol significantly increased during the high cholesterol diet infusion while that of endogenous cholesterol decreased. This decrease or compensation in endogenous cholesterol transport occurred during absorption of increased levels of dietary cholesterol and apparently was due to an approximate 50% decrease in the absorption of lumenal cholesterol. Our data demonstrated that there was not a simple one-to-one relationship between the amount of isotopic dietary cholesterol absorbed from the intestinal lumen and the mass of cholesterol moved into lymph via the intestine. Rather, the amount of sterol transported into the body depends on the degree of the decrease in endogenous cholesterol transport and probably on the efficiency of cholesterol esterification during absorption.

Animals↗
PubMed1983

Effect of dietary cholesterol level on the composition of thoracic duct lymph lipoproteins isolated from nonhuman primates.

The effect of two different levels of dietary cholesterol (0.16 mg/Kcal and 0.79 mg/cal) on the composition of thoracic lymph duct lipoproteins was studied in two species of nonhuman primates, Ceropithecus aethiops (African green monkey) and Macaca fascicularis (cynomolgus monkey). Diet was infused intraduodenally at a constant rate to facilitate comparisons among animals. The higher level of dietary cholesterol resulted in an increase in the amount of cholesteryl ester in lymph chylomicrons and VLDL. Cholesteryl oleate was the predominant cholesteryl ester present in lymph d less than 1.006 g/ml lipoproteins and it was the predominant cholesteryl ester formed from exogenous radiolabeled cholesterol. The percentage of saturated and monounsaturated cholesteryl esters in lymph chylomicrons and VLDL significantly increased with the higher dietary cholesterol level. The apoprotein distribution of chylomicrons and VLDL was qualitatively similar during infusions of both diets. The apoprotein B of intestinal chylomicrons and VLDL, termed apoprotein B2, was qualitatively similar during low and high cholesterol diet infusion and was significantly smaller than that of plasma LDL apoB, termed apoprotein B1, as indicated by its electrophoretic mobility in SDS-polyacrylamide gels. The major phospholipid present in lymph chylomicrons and VLDL was phosphatidylcholine and the phospholipid composition of the particles was not affected by diet. Lymph d greater than 1.006 g/ml lipoproteins were separated and the cholesterol mass distribution among lipoprotein fractions was found to be similar during both diet infusions. With an increase in the level of dietary cholesterol, the percentage esterification of cholesterol mass and of exogenous cholesterol radioactivity increased in LDL and HDL from lymph. Lymph LDL and HDL contained less free and esterified cholesterol when their composition was compared to that for these lipoproteins in plasma. We conclude that the primary effect of increased dietary cholesterol level was to increase the cholesteryl ester content of all lymph lipoproteins; cholesterol distribution among lymph lipoproteins was unaffected.

Animals↗
PubMed1982

Opioid peptides and noradrenaline co-exist in large dense-cored vesicles from sympathetic nerve.

The possibility that opioid peptides and noradrenaline co-exist not only in the desheathed bundle of bovine splenic nerve which contains approximately 98% sympathetic C-fibers, but also in the population of large dense-cored noradrenergic vesicles from these fibers, has been investigated. The primary fraction of large dense-cored vesicles which can be prepared at about 85% purity has been further subjected to density gradient and fractional centrifugation procedures, including D2O-loading and unloading on modified second gradients, in an attempt to separate any minor population of particles which potentially could contain opioid peptides and contaminate the large dense-cored vesicle fraction. Measurement of opioid peptides, noradrenaline, dopamine and dopamine beta-hydroxylase activity supports the conclusion that opioid peptides are stored in the primary population of large dense-cord vesicles per se, rather than in a minor population of contaminating particles from cells other than sympathetic C-fibers. This conclusion has implications for exocytotic release and the physiological role of the opioid peptides intra- and extra-neuronally. Nerve vesicle opioid peptides have a size less than 5000 daltons, in contrast to the high proportion of large peptides containing enkephalin sequences in the bovine adrenal medulla.

Adrenergic Fibers↗
PubMed1980

Idiopathic orthostatic hypotension: circulating noradrenaline and ultrastructure of saphenous vein.

The present study indicates that patients who can be clinically classified as idiopathic orthostatic hypotensives of the peripheral type are heterogeneous. There is the typical hypoadrenergic type characterized by low levels of circulating noradrenaline, much reduced or absent noradrenaline stores, and correspondingly little or no adrenergic innervation in the saphenous vein, a major capacitance vessel, as confirmed by ultrastructural examination. In one patient of this type, an abnormally high occurrence of mast cells in the blood vessel wall was noted. There also exists a category of individuals of the hyperadrenergic type, analogous to certain diabetics with noradrenergic abnormalities. These patients also are characterized by low levels of circulating noradrenaline, but noradrenaline stores are high; an exaggerated release of neurotransmitter occurs in response to stimuli; the saphenous vein with noradrenergic innervation remains ultrastructurally normal; however, the effector cell response are greatly blunted. In one patient of this type, the smooth muscle cells of the saphenous vein contained excessively high glycogen deposits. Further, it should be anticipated that a variety of intermediate types can be found as exemplified by one patient with variable, low to normal levels of circulating noradrenaline, a sluggish response to release neurotransmitter upon postural challenge, and considerable innervation of saphenous vein but with the majority of axons and terminals undergoing active degeneration.

Adrenergic Fibers↗
PubMed1979

Dopamine beta-hydroxylase distribution in density gradients: physiological and artefactual implications.

Knowledge of the vesicular origin of circulating dopamine beta-hydroxylase (DbetaH) is indispensable for any attempts to explain the parallelism or lack of it between circulating enzyme and catecholamines as they may relate to physiological stress, forms of hypertension, neurological disorders, and the response to pharmacological agents. The present study represents an effort to evaluate and to place in proper perspective data based on the DbetaH activity found in the region of the light vesicle peak of noradrenaline (NA), which is used as a quantitative measure of a population of small terminal vesicles. Distributions of vesicles and subvesicular components are compared with DbetaH and NA in sucrose-D2O density gradients used to prepare relatively pure fractions of large dense cored vesicles (LDV) from bovine splenic nerve. Although NA in sedimentable particles of the light vesicle peak is likely to be a valid measure of a small vesicle population, the following is demonstrated: (1) A substantial fraction (25%-37%) of the total sedimentable DbetaH activity can be proven to distribute in the region of the light vesicle peak from a tissue with an insignificant small vesicle population. Based on studies of vesicles from sequential nerve segments, this enzyme activity probably corresponds to a population of "immature" LDV which are undergoing axoplasmic transport and have not synthesized their full complement of transmitter. (2) Physical lysis which depletes the matrix of LDV causes redistribution of DbetaH activity from the heavy vesicle peak into the region of the light vesicle peak. Analogously, DbetaH associated with exocytosed LDV and retrograde transport particles is also likely to contaminate the region of the light vesicle peak. (3) Based on available data, it can be calculated that each small dense cored vesicle could contain only 0.1-0.5 molecules of DbetaH and that a contamination of only 0.016% LDV can account for all of the DbetaH reported to occur in the light vesicle peak of normal rat vas deferens preparations.

Animals↗
PubMed1979

Catecholamine-rich cells and varicosities in bovine splenic nerve, vesicle contents and evidence for exocytosis.

The bovine splenic nerve trunk contains mast cells, ganglion cells, small intensely fluorescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structures could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300-800 nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120-140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45-55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicles in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.

Animals↗
PubMed1976

Norepinephrine:adenosinetriphosphate ratios in purified adrenergic vesicles.

Norepinephrine (NE):adenosinetriphosphate (ATP) ratios were studied in a highly purified fraction of large dense core vesicles isolated from the bovine splenic nerve. Vesicles prepared from nerves chilled approximately 10 and 30 min post mortem were compared. The NE:ATP molar ratio decreased from 6.3 to 4.8, p less than 0.005; NE decreased from 61 to 42 nmol, while ATP decreased only from 9.6 to 8.8 nmol/mg protein. Animals weighing 180-360 kg were compared with heavier ones weighing 400-700 kg. NE increased from 42 to 68 nmol and ATP increased from 5.9 to 13.2 nmol/mg protein, while the NE:ATP molar ratio decreased from 7.2 to 5.2, p less than 0.005. Changes during vesicle maturation were studied by comparing vesicles identically prepared from equal weights of a proximal nerve segment close to the coeliac ganglion and a distal, intrasplenic segment. NE increased from 45 to 70 nmol while ATP remained unchanged at 10.0 nmol/mg protein and the NE:ATP molar ratio increased from 4.5 to 7.0, p less than 0.005. It was interpreted that vesicle ATP content, like dopamine beta-hydroxylase, was established early in the cell body and remained unchanged during axoplasmic transport. ATP was in a complex which was relatively stable to post mortem hydrolysis at least between 10 and 30 min prior to chilling the nerves. The addition of newly synthesized NE into a readily releasable pool during axoplasmic transport occurs without ATP and can account for the increased ratio above 4:1 in the distal segment vesicles.

Adenosine Triphosphate↗