Joseph Eichberg

Determination of protein by a modified Lowry procedure in the presence of some commonly used detergents.

A wide variety of detergents has been employed to solubilize and purify membrane proteins (1,2). The interference caused by certain cationic and nonionic detergents in the Lowry procedure for protein determination (3) and its elimination by inclusion of sodium dodecyl sulfate (SDS)l has been previously described (4-7). However, only for Triton X-100 has this modification been shown definitively to be effective at concentrations of interfering detergent as high as 1% in the final reaction mixture over a range of protein concentrations (45). In this communication, we further document the applicability of a modified Lowry method to protein preparations containing high levels of nonionic and cationic detergents in common use.

Metabolism of Phospholipids in Peripheral Nerve from Rats with Chronic Streptozotocin‐induced Diabetes Increased Turnover of Phosphatidylinositol‐4,5‐Bisphosphate

Abstract: The effect of chronic streptozotocin‐induced diabetes on phospholipid metabolism in rat sciatic nerve in vitro was investigated. In normal nerve incubated for 2 h in Krebs‐Ringer‐bicarbonate buffer containing [32P]orthophosphate, radioactivity was primarily incorporated into phosphati‐dylinositol‐4,5‐bisphosphate and phosphatidylcholine. Smaller amounts were present in phosphatidylinositol‐4‐phosphate, phosphatidylinositol, and phosphatide acid. As compared to controls, phosphatidylinositol‐4,5‐bisphosphate in nerves from animals made diabetic 2, 10, and 20 weeks earlier accounted for 30–46% more of the isotope, expressed as a percentage, incorporated into all phospholipids. In contrast, the proportion of radioactivity in phosphatidylcholine decreased by 10–25%. When the results were expressed as the quantity of phosphorus incorporated into phospholipid, only phosphatidylinositol‐4,5‐bisphosphate displayed a change. The amount of isotope which entered this lipid increased 60% and 67% for 2‐ and 10‐week diabetic animals, respectively. Increased phosphatidylinositol‐4,5‐bisphosphate labeling was observed when epineurial‐free preparations were used or when the composition of the incubation medium was varied. Sciatic and caudal nerve conduction velocities were decreased after 10 and 20 weeks but were unchanged after 2 weeks. We conclude that an increase in the turnover of phosphatidylinositol‐4,5‐bisphosphate in sciatic nerve from streptozotocin‐diabetic rats appears relatively early and persists throughout the course of the disease. This metabolic alteration may be related to a primary, defect responsible for the accompanying deficient peripheral nerve function.

Sphingomyelin Functions as a Novel Receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.

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

Abstract: 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.

Protein kinase C changes in diabetes: Is the concept relevant to neuropathy?

Protein kinase C (PKC) comprises a superfamily of isoenzymes, many of which are activated by 1,2-diacylglycerol (DAG) in the presence of phosphatidylserine. In order to be capable of DAG activation, PKC must first undergo a series of phosphorylation at three conserved sites. PKC isoforms phosphorylate a wide variety of intracellular target proteins and have multiple functions in signal transduction-mediated cellular regulation. An elevation in DAG levels and an increase in composite PKC activity and/or certain isoforms occurs in several nonneural tissues from diabetic animals, including the vasculature. The ability of isoform-specific PKC inhibitors to antagonize diabetes-induced abnormalities has implicated altered PKC beta activity in the onset of several diabetic complications, In contrast to many other tissues, DAG levels fall in diabetic nerve and a consistent pattern of change in PKC activity has not been observed. Treatments that alter PKC activity affect nerve Na+, K+-ATPase activity, but the mechanism involved is not well understood, Inhibition of PKC beta in diabetic rats appears to correct reduced nerve blood flow and decreased nerve conduction velocity. These and other findings indicate that changes in the neurovasculature exert adverse effects during the pathogenesis of diabetic neuropathy. Still unresolved is a clear-cut role for PKC in the development of abnormalities in neural cell metabolism. Further progress will depend on a more complete understanding of the functions of individual PKC isoforms in nerve. Future investigation could focus profitably on biochemical processes in nerve cells that modulate PKC activity and that are altered in diabetes, such as vascular endothelial growth factor levels and production of reactive oxygen species arising from oxidative stress.

Myelin P0: New Knowledge and New Roles

Protein zero (P0) is an integral transmembrane glycoprotein that serves as the major protein component of peripheral nerve myelin and is a member of the immunoglobulin (IgG) gene superfamily. As a cell adhesion molecule, P0 mediates homophilic adhesive interactions between Schwann cell plasma membranes and is a key structural constituent of both the major dense line and intraperiod line of compact myelin. Both the extracellular and cytoplasmic domains contribute to these interactions and evidence indicates that the post-translational modifications of the molecule, including glycosylation, acylation and phosphorylation, play an important modulatory role in adhesion and likely in the proper trafficking of P0 from the endoplasmic reticulum to the plasma membrane as well. Structural and genetic studies indicate that mutations in P0 producing human demyelinating diseases probably do so by perturbing or preventing homophilic interactions during myelination, or by producing cellular toxicity or an unstable myelin sheath. A variety of transcription factors, growth factors and neurosteroids both directly and indirectly influence P0 gene expression during maturation of the myelinating Schwann cell. Besides its structural function in myelin, P0 may have roles in the delivery of other Schwann cell proteins to their proper location, especially at or near nodes of Ranvier, and in neuronal-glial interactions.

Phosphorylation of myelin proteins: Recent advances

Since it was first described 25 years ago, phosphorylation has come to be recognized as a widespread and dynamic post-translational modification of myelin protein. In this review, the phosphorylation characteristics of myelin basic protein, protein zero (P0), myelin-associated glycoprotein and 2′3′ cyclic nucleotide 3′-phosphodiesterase are summarized. Emphasis is placed on recent advances in our knowledge concerning the protein kinases involved and the sites, of phosphorylation in the amino acid sequences, where known. The possible roles of myelin protein phosphorylation in modulating myelin structure, the process of myelin assembly and mediation of signal transduction events are discussed.

Alterations in retinal Na+, K+ATPase in diabetes : streptozotocin-induced and Zucker diabetic fatty rats

The temporal pattern of changes in the specific activities of retinal Na+, K(+)-ATPase (Na, K-ATPase) and Mg(2+)-ATPase (Mg-ATPase) were determined at several time intervals following the onset of diabetes in streptozotocin-induced diabetic (STZ: at 1, 2, 4 and 6 months) Long-Evans hooded rats, spontaneously diabetic Zucker diabetic fatty (ZDF: at 1, 2 and 4 months) rats and their age-matched controls. These animals were utilized as models for insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), respectively. Na, K-ATPase specific activity, using 10(3) M ouabain, was decreased (-6% to -14%) at all time points after the appearance of hyperglycemia in the ZDF rat, but was reduced only after 4 and 6 months in the STZ rat (-8% and -14%, respectively). In contrast, Mg-ATPase activity was significantly increased (13%) after 4 months in the ZDF rat and after 6 months in the STZ rat (8%). The concentration-dependent inhibitory effects of ouabain (10(-9) to 10(-3) M) on the activity of Na, K-ATPase in diabetic rats and age-matched controls was used to assess the time-dependent effects of diabetes on the alpha 3-high ouabain affinity or the alpha 1-low ouabain affinity retinal Na, K-ATPase isozymes. The retinal Na, K-ATPase activity for the alpha 3 isozyme was significantly lower at all times examined for the ZDF (-5% to -26%) and STZ-induced diabetic rats (-8% to -14%). This was reflected in the markedly decreased half-maximal inhibitory concentrations (IC50) of ouabain for the alpha 3 isozyme. For example, after four months of diabetes, the mean +/- SEM IC50 values were 12 +/- 3 nM in the STZ rats and 48 +/- 6 nM in the age-matched controls and 19 +/- 3 nM in the ZDF rats and 30 +/- 4 nM in the age-matched controls. In contrast, the activity of the alpha 1 isozyme was slightly, but significantly, decreased at 2 and 4 months in the ZDF rats (-4% to -7%) and after 4 and 6 months in the STZ-induced diabetic rats (-3% to -9%) while the IC50 values were unchanged. Moreover, the Hill coefficient for the alpha 3 isozyme was decreased in both diabetic groups while it was unchanged for the alpha 1 isozyme.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Abstract: 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, my‐elin, 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 subcellu‐lar 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.

Purification of Phosphatidylinositol Synthetase from Rat Brain by CDP‐Diacylglycerol Affinity Chromatography and Properties of the Purified Enzyme

A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP‐1,2‐diacylsn‐glycerol:myo‐inositol 3‐phosphatidyltransferase; EC 2.7.8.11) is described. The enzyme was purified 200–250‐fold from the homogenate by solubilization with Triton X‐100 from microsomal membranes and affinity chromatography on CDP‐diacylglycerol‐Sepharose. Elution of enzyme activity required the presence of Triton X‐100, CDP‐diacylglycerol, and either phosphatidylcholine or asolectin. The product that was obtained in 5–10% yield from whole brain and in 70% yield from the microsomal fraction contained three protein bands as determined by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The final preparation contained levels of CDP‐diacylglycerol hydrolase and CDP‐diacylglycerol: sn‐glycero‐3‐phosphate 3‐phosphatidyltransferase activities that were less than 1% of PI synthetase activity. The purified enzyme displayed a pH optimum of 8.5–9.0, required either Mg2+ or Mn2+ and exhibited a Km of 4.6 mM for myo‐inositol.