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Biomedical subjects

J Eichberg

Publications and source records attributed to J Eichberg.

At least 55 records · Page 3Linked to original sources
PubMed1990

A myo-inositol pool utilized for phosphatidylinositol synthesis is depleted in sciatic nerve from rats with streptozotocin-induced diabetes.

Peripheral nerve from experimentally diabetic rats exhibits lowered levels of myo-inositol (MI) and decreased incorporation of [3H]MI into phosphatidylinositol (PI). There are indications that diminished PI turnover may be causally related to reduced Na+,K(+)-ATPase activity in diabetic nerve. We have investigated whether a metabolic compartment of MI that is essential for PI synthesis is decreased in this tissue. Sciatic nerve segments from streptozotocin-induced diabetic and age-matched normal rats were incubated in vitro with either 32Pi or [3H]cytidine in the presence of propranolol. This cationic amphiphilic agent redirected nerve phospholipid metabolism to produce enhanced 32P incorporation into PI and decreased labeling of phosphatidylcholine and phosphatidyl-ethanolamine. The accumulation of phosphatidyl CMP (CMP-PA) was also demonstrated by chromatographic and enzymatic means. The incorporation of [3H]cytidine into CMP-PA in normal nerve increased up to 15-fold when 0.6 mM propranolol was present. In diabetic nerve, the liponucleotide incorporated 2- to 3-fold more isotope and was more readily labeled at lower drug concentrations as compared to normal nerve. The buildup of [3H]CMP-PA was reduced in a dose-dependent manner in the presence of MI in the incubation medium at concentrations up to 3 mM. However, if MI was added after liponucleotide accumulation, preformed CMP-PA could not be utilized for PI synthesis. The difference in liponucleotide labeling between normal and diabetic nerve was nearly abolished at 0.3 mM medium MI, a concentration much less than the level of cyclitol in the tissue. These results strongly suggest the presence in nerve of a pool of MI that is not in equilibrium with the bulk of nerve MI and that is preferentially used for PI synthesis. This metabolic compartment is depleted in diabetic nerve but can be readily replenished by exogenous MI and may correspond to the MI pool that has been proposed to be required for the turnover of a portion of tissue PI involved in maintenance of normal Na+,K(+)-ATPase activity.

Animals↗
PubMed1990

1,2-diacylglycerol content and its arachidonyl-containing molecular species are reduced in sciatic nerve from streptozotocin-induced diabetic rats.

The content of 1,2-diacylglycerol (DAG) was determined in sciatic nerves from normal and streptozotocin-induced diabetic rats. In nerves frozen in situ, DAG content was reduced 22% in the proximal region and 77% in the distal region of diabetic nerve, principally because of the loss of associated fat. DAG levels in freshly dissected and desheathed diabetic nerve were decreased from 23 to 30% as compared with normal nerve. Determination of DAG molecular species distribution in desheathed normal nerve indicated that 18:0/20:4 accounted for 34%, 16:0/18:1 for 17%, and several other polyunsaturated fatty acid-containing species for 17% of the total. In diabetic nerve, the quantity of the 18:0/20:4 DAG, species was reduced by 37%, and this drop was 62% of the reduction in all molecular species. The content of the minor species, 16:0/20:4 DAG, was decreased by 48%. Our results suggest that nerve DAG arises in large part from phosphoinositide degradation. Moreover, these results provide support for the hypothesis that reduced Na+,K(+)-ATPase activity in diabetic nerve is a consequence of decreased phosphoinositide turnover, which thereby generates insufficient DAG to maintain a protein kinase C-mediated step necessary for activation of Na+,K(+)-ATPase.

Animals↗
PubMed1990

Distribution of elements and water in peripheral nerve of streptozocin-induced diabetic rats.

Accumulating evidence suggests that alterations in Na, Ca, K, and other biologically relevant elements play a role in the mechanism of cell injury. The pathogenesis of experimental diabetic neuropathy is unknown but might include changes in the distribution of these elements in morphological compartments. In this study, this possibility was examined via electron-probe X-ray microanalysis to measure both concentrations of elements (millimoles of element per kilogram dry or wet weight) and cell water content (percent water) in frozen, unfixed, unstained sections of peripheral nerve from control and streptozocin-induced diabetic rats. Our results indicate that after 20 wk of experimental diabetes, mitochondria and axoplasm from myelinated axons of proximal sciatic nerve displayed diminished K and Cl content, whereas in tibial nerve, the intraaxonal levels of these elements increased. In distal sciatic nerve, mitochondrial and axoplasmic levels of Ca were increased, whereas other elemental alterations were not observed. These regional changes resulted in a reversal of the decreasing proximodistal concentration gradients for K and Cl, which exist in nondiabetic rat sciatic nerve. Our results cannot be explained on the basis of altered water. Highly distinctive changes in elemental distribution observed might be a critical component of the neurotoxic mechanism underlying diabetic neuropathy.

Animals↗
PubMed1990

Correction of altered metabolic activities in sciatic nerves of streptozocin-induced diabetic rats. Effect of ganglioside treatment.

The effect of ganglioside administration to nondiabetic and streptozocin-induced diabetic rats on sciatic nerve Na(+)-K(+)-ATPase, polyphosphoinositide (PPI) turnover, and protein phosphorylation was investigated. Gangliosides were injected (10 mg/kg body wt i.p.) for 10 or 30 days beginning 20 days after induction of diabetes. Na(+)-K(+)-ATPase activity was reduced nearly 50% in diabetic nerve and was restored to normal by both ganglioside treatments. The elevated levels of fructose and sorbitol and depressed content of myoinositol in diabetic nerve were unaffected by 30 days of ganglioside treatment, indicating that the restoration of Na(+)-K(+)-ATPase activity is not dependent on normal concentrations of these compounds. In the same nerves, 32P incorporation into phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate increased 73-76 and 39-53%, respectively, in diabetic compared with nondiabetic tissue. Ganglioside administration abolished the elevated labeling of PPIs after 30 days but was ineffective after only 10 days. Neither ganglioside regimen was able to reverse enhanced phosphorylation of the major peripheral nerve myelin protein P0. The finding that gangliosides can more quickly correct the effects of diabetes on Na(+)-K(+)-ATPase activity than on PPI turnover suggests that the mechanisms underlying these two phenomena are not closely related and are distinct from the sequence of events responsible for altered myelin protein phosphorylation.

Animals↗
PubMed1989

Interactions of thiophosphatidic acid with enzymes which metabolize phosphatidic acid. Inhibition of phosphatidic acid phosphatase and utilization by CDP-diacylglycerol synthase.

Thiophosphatidic acid (1,2-diacyl-sn-glycero-3-phosphorothioate; thioPA) was chemically synthesized from egg phosphatidylcholine-derived 1,2-diacylglycerol and PSCl3 and tested for its effects on enzymes which utilize phosphatidic acid (PA) in phospholipid biosynthesis. The compound was not a substrate for rat liver cytosolic PA phosphatase and strongly inhibited this enzyme activity. ThioPA was also a potent inhibitor of purified membrane-associated PA phosphatase from Saccharomyces cerevisiae in a competitive manner and exhibited an apparent Ki = 60 microM. In contrast, purified CDPdiacylglycerol synthase (PA:CTP cytidylyltransferase) from this organism was able to convert thioPA to CDP-diacylglycerol. The apparent Vmax for thioPA was 7-fold lower than that for PA, whereas the apparent Km for thioPA (70 microM) was 4-fold lower than that for PA. Calculation of the specificity constant (Vmax/Km) demonstrated that PA was the preferred substrate. These properties of thioPA indicate that this substance may prove useful in studies of phospholipid metabolism and function.

Animals↗
PubMed1989

Hexanedione effects on protein phosphorylation in rat peripheral nerve.

Rats were treated with either 2,5-hexanedione (2,5-HD), 1,6-hexanediol (1,6-HDIOL), or saline for 7, 15 or 24 days. Protein phosphorylation was measured in proximal and distal sciatic nerve segments following incubation with [32P]orthophosphate. In proximal segments, 2,5-HD administration caused selective time-dependent increases in isotope incorporation in a 55 kDa protein, tentatively identified as tubulin, and a 180 kDa protein. Enhanced phosphorylation was highest at 24 days when motor function was most impaired. Administration of 1,6-HDIOL produced no consistent phosphorylation changes. Animals intoxicated with 3,4-dimethyl-2,5-hexanedione for 12 days showed proximal region increases in phosphorylation of the 55 and 180 kDa proteins and the major myelin proteins, Po and Pr.

Animals↗
PubMed1989

Protective effect of a synthetic peptide comprising the complete preS2 region of the hepatitis B virus surface protein.

A peptide was synthesized containing the entire 55 amino acid residue sequence of the hepatitis B virus (HBV) surface antigen preS2 region (ad subtype). The unconjugated peptide was inoculated into four chimpanzees. Following multiple injections, all of the animals developed specific antipeptide antibodies that reacted with intact surface antigen particles bearing the preS2 moiety. All four peptide-inoculated animals were found to be protected from infection after intravenous challenge with live HBV of either the ad or ay subtypes.

Animals↗
PubMed1989

Relationship of ATP turnover, polyphosphoinositide metabolism, and protein phosphorylation in sciatic nerve and derived peripheral myelin subfractions from normal and streptozotocin diabetic rats.

Sciatic nerve from streptozotocin-induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [gamma-32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P]orthophosphate for 2 h, the specific activity of PIP2 was 50-120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P-labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a "nonmyelin" fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin-enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.

Adenosine Triphosphate↗
PubMed1989

Alteration of phosphoinositide metabolism, protein phosphorylation, and carbohydrate levels in sciatic nerve from Wistar fatty diabetic rats.

Sciatic nerve from the Wistar fatty diabetic (FD) rat, a prospective model for non-insulin-dependent diabetes mellitus, was investigated to determine the content of carbohydrates and to measure the incorporation of 32P into phosphoinositides and proteins. This strain has been shown to develop structural abnormalities in nerves and to exhibit reduced conduction velocity. Males became diabetic between the ages of 8 and 10 wk and were maintained together with lean sibling controls until the animals were either 22 or 44 wk old. Throughout this period, FD rats displayed moderate hyperglycemia. The carbohydrate profile of FD rat sciatic nerve exhibited modest increases in glucose, fructose, and sorbitol levels and significantly reduced myo-inositol concentrations, which were comparable at both ages. When nerves from 22-wk-old animals were incubated with [32P]orthophosphate and incorporation of radioactivity into phospholipids was measured, an increase in isotope uptake into phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-4-phosphate in the distal portions of tissue from the FD rat was observed. This effect was more pronounced in nerves from 44-wk-old rats. Phosphorylation of the major myelin protein P0 was 70% higher in the most distal portion of FD sciatic nerve from 22-wk-old animals. A comparable rise in phosphorylation of P0 as well as the large (P1) and small (Pr) myelin basic proteins occurred in nerves from 44-wk-old rats. In these animals, an approximately 50% decrease in the uptake of 32P into P0 and P1 in the most proximal region of FD nerve was also apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Distribution of elements in rat peripheral axons and nerve cell bodies determined by x-ray microprobe analysis.

X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.

Animals↗
PubMed1988

Phospholipid metabolism and protein phosphorylation in sciatic nerve from genetically diabetic (db/db) mouse.

The incorporation of [32P]orthophosphate into phospholipids and proteins of sciatic nerve from genetically diabetic (db/db) and littermate control (db/m) C57BL/KsJ mice was studied. Nerves from animals of ages 12, 16, 22, 26, and 38 wk were incubated in vitro. Among phospholipids, the uptake of isotope into phosphatidic acid was higher at nearly all ages examined. Phosphorylation of several proteins, including the major myelin glycoprotein, P0, and the small myelin basic proteins Pr + P2, was significantly enhanced in nerves from both 12- and 38-wk-old diabetic mice. The altered pattern of protein phosphorylation, but not that of phospholipid metabolism, was similar to changes observed in sciatic nerve from streptozocin-induced diabetic rats. The relationship of the results to reported levels of myo-inositol, sorbitol, and Na+-K+-ATPase activity and to functional abnormalities in nerves of db/db mice is discussed. The findings suggest that caution should be exercised in reaching conclusions concerning which biochemical alterations observed in different animal models of diabetic neuropathy are invariably associated with the development of this disorder.

Animals↗
PubMed1987

Synthesis of phosphatidylethanolamine under possible primitive earth conditions.

The synthesis of phosphatidylethanolamine was accomplished when a mixture of phosphatidic acid, ethanolamine, and cyanamide at pH 7.3 was taken to dryness and heated at temperatures ranging from 25 to 60 degrees C for 6 h. Chromatographic, enzymatic, and chemical techniques were used to identify and confirm that phosphatidylethanolamine had been formed. This work indicates that the synthesis of this compound can occur starting with precursors and conditions that are presumed to have existed on the primitive Earth.

Carbon Radioisotopes↗
PubMed1987

Fatty acid patterns in triglycerides, diglycerides, free fatty acids, cholesteryl esters and phosphatidylcholine in serum from vegetarians and non-vegetarians.

The differences in the fatty acid spectra of serum samples obtained from vegetarians (62 females, 40 males) and non-vegetarians (70 females, 38 males) were evaluated in a matched-pair study design. This study population made it possible to examine 48 female and 31 male pairs whose age difference did not exceed 3 years. The pairs were further matched by education, social status and health-consciousness. The fatty acid pattern of whole serum total lipids and HDL total lipids were determined by GLC. In particular linoleic, linolenic, oleic and docosahexaenoic acid reveal statistically significant differences due to different nutritional habits. A subsample (n = 20) of sera from the 2 groups was investigated by separation of lipid classes by TLC and GLC on a SP 2,340 fused-silica capillary column in order to separate cis-trans fatty acids additionally. This part of the study gives detailed information concerning the fatty acid composition of cholesteryl esters, triglycerides, diglycerides, free fatty acids and phosphatidylcholine. In all those fractions the fatty acid profiles reflect the dietary consumption of lipids. Palmitoleic, vaccenic and docosahexaenoic acid as markers of omnivorous nutrition reach levels of 5, 5 and 3% respectively in non-vegetarians, while they remain remarkably lower in vegetarians. The most prominent difference is the higher amount of linoleic acid in all lipid classes of vegetarian serum samples. The highest amount of trans fatty acids (up to 3%) was detected in di- and triglycerides.

Adolescent↗
PubMed1987

Altered protein phosphorylation in sciatic nerve from rats with streptozocin-induced diabetes.

The effect of experimental diabetes on the phosphorylation of proteins in the rat sciatic nerve was studied. Nerves from animals made diabetic with streptozocin were incubated in vitro with [32P]orthophosphate and divided into segments from the proximal to the distal end, and proteins from each segment were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The principal labeled species were the major myelin proteins, P0, and the basic proteins. After 6 wk of diabetes, the incorporation of isotope into these proteins rose as a function of distance along the nerve in a proximal to distal direction and was significantly higher at the distal end compared with incorporation into nerves from age-matched controls. The overall level of isotope uptake was similar in nerves from diabetic animals and weight-matched controls. The distribution of 32P among proteins also differed in diabetic nerve compared with both control groups in that P0 and the small basic protein accounted for a greater proportion of total label incorporated along the entire length of nerve. In contrast to intact nerve, there was no significant difference in protein phosphorylation when homogenates from normal and diabetic nerve were incubated with [32P]-gamma-ATP. The results suggest that abnormal protein phosphorylation, particularly of myelin proteins, is a feature of experimental diabetic neuropathy and that the changes are most pronounced in the distal portion of the nerve.

Animals↗
PubMed1986

Dioctanoylglycerol and phorbol diesters enhance phosphorylation of phosphoprotein B-50 in native synaptic plasma membranes.

The short chain diacylglycerol, 1,2-dioctanoylglycerol, at concentrations of 100-300 microM stimulated phosphorylation of the nervous system-specific membrane protein B-50 (Mr 48 kDa, IEP 4.5) in isolated synaptic plasma membranes both in the presence and absence of exogenous protein kinase C. Comparable enhancement of histone phosphorylation by purified protein kinase C was achieved with 1 microM neutral lipid. Phorbol dibutyrate was 100 times more potent than the diacylglycerol in stimulating endogenous B-50 kinase in the membranes, whereas 4-alpha-phorbol was without effect. These results further confirm that B-50 is phosphorylated physiologically by a C kinase. Our data are consistent with a negative feedback mechanism in which generation of 1,2-diacylglycerol by enhanced phosphatidylinositol-4,5-bisphosphate hydrolysis could stimulate B-50 phosphorylation, thereby diminishing phosphatidylinositol-4-phosphate kinase activity and decreasing phosphatidylinositol-4,5-bisphosphate biosynthesis.

Animals↗
PubMed1986

Insulin reverses enhanced incorporation of 32P into polyphosphoinositides in peripheral nerve of the streptozotocin diabetic rat.

The ability of insulin treatment to reverse altered phosphoinositide metabolism in sciatic nerve from streptozotocin diabetic rats was studied. Diabetes was induced in rats by means of a single injection of streptozotocin. Enhanced incorporation of 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) was detectable as early as 8 days following intravenous injection of streptozotocin and was maximal after 4 weeks. Hormone treatment was initiated at this time by daily injections of protamine zinc insulin followed by the implantation of long-acting insulin osmotic minipumps, and 4 weeks later sciatic nerves were removed and incubated in the presence of [32P]orthophosphate. The increased labeling of PIP2 was completely reversed by hormone administration. In contrast, insulin (0.1 and 1.0 mU/ml) added to the incubation medium failed to reverse the altered pattern of 32P incorporation into PIP2. The uptake of 32P into PIP2 was greater than 80% higher into the proximal than into the distal portion of normal sciatic nerve when these were incubated separately. This metabolic difference was abolished in diabetic rats, although the incorporation into both segments was still significantly higher than in controls. These results strengthen the association of altered nerve PIP2 metabolism with the diabetic state and are consistent with the concept that experimental diabetic neuropathy is a distal axonopathy.

Animals↗