Biosynthesis of gangliosides in primary cultures of rat hepatocytes. Determination of the net synthesis of individual gangliosides by incorporation of labeled N-acetylmannosamine

The ganglioside content of rat hepatocytes increases several-fold during the first 6 days in monolayer culture. To correlate increased levels with rates of de novo synthesis, the incorporation of N-acetyl-[6-3H]D-mannosamine into individual gangliosides was determined. The calculation of synthetic rates was made possible by the simultaneous measurement of the specific radioactivity of the immediate sialic-acid donor, CMP-Neu5Ac. The CMP-Neu5Ac content of hepatocytes was found by HPLC analysis to be 30.5 nmol/g of plated cells. The specific radioactivity of this precursor pool reached a constant plateau 5 h after addition of the labeled N-acetyl-mannosamine and remained constant for at least 70 h. The incorporation into individual gangliosides was measured in primary cultures of rat hepatocytes between 72 and 144 h after seeding. During this period, the increase in ganglioside levels was greatest. The highest rates of incorporation were seen in GD1a followed by GM3, GM1, GD3 and the polysialylated compounds. The following rates of synthesis (nmol per 60 h and mg of protein) were calculated: GD1a 0.68, GM3 0.59, GM1 0.36, GD3 0.13 and GT1 0.02. These values are compared with the net increase of the gangliosides as measured by the resorcinol reaction.     

HDAC6 inhibition restores TDP‐43 pathology and axonal transport defects in human motor neurons with TARDBP mutations

TDP‐43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP‐43, are one of the common causes of familial ALS. In this study, we investigate TDP‐43 protein behavior in induced pluripotent stem cell (iPSC)‐derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP‐43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP‐43, C‐terminal fragments, and phospho‐TDP‐43. By generating iPSC lines with allele‐specific tagging of TDP‐43, we find that mutant TDP‐43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP‐43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP‐43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP‐43 pathology.     

Positive cooperativity in binding carbon monoxide to hemocyanin

The binding of carbon monoxide to hemocyanin from the crab $\text{Scylla serrata}$ has been studied by thin layer optical absorption and front face fluorescence techniques. The binding to the monomeric form is completely noncooperative whereas the binding to the native oligomeric form is found to be weakly but definitely cooperative. An analysis based on the MWC model of the oxygen and carbon monoxide binding curves indicates that the allosteric constant, L, describing the equilibrium between the 2 unligated forms is different for each ligand. This implies that at least 3 allosteric forms are needed to characterize the binding of oxygen and carbon monoxide to this hemocyanin.     

Integrin on developing and adult skeletal muscle

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu     

Seasonal characteristics of and age at menarche

Data from approximately 600 U.S. women were analyzed for seasonality in menarche. Data were converted to the ratio of observed (O) to expected (E) cases, based on an equal distribution of events over the year, per considered time span of the year. The greatest O/E ratio in menarche (1.55) occurred during January, followed by August (1.43) and July (1.24). The lowest O/E ratio in menarche occurred in February and May (0.68). Seasonal patterns in menarche were detected for the separate cohorts of women born during the 1940s, 1950s, and 1960s, with a shift from a December-January peak in menarche for those born in the 1940s and 1950s to an August-September peak for those born in the 1960s. Girls who were younger (8-14 yrs) at menarche exhibited a seasonal difference in the peak number of menarches by about 6 months in comparison to the pattern for girls who were older (15-17 yrs). Girls who experienced menarche during August-October were statistically significantly younger (p less than 0.05) than those born during the other three seasons. Season of birth was not statistically significantly associated with season of menarche. Overall, no 12-month pattern was substantiated by cosinor analysis in the month of menarche. However, a 6-month rhythm was detected in menarche for women born between 1940 and 1960 (p = 0.004, A = 29% M, phi = January and July).     

Signal transduction in endotoxin-stimulated synthesis of TNF-alpha and prostaglandin E2 by rat Kupffer cells. Role of extracellular calcium ions and protein kinase C.

Endotoxin is a well established elicitor of cytokine production in mononuclear cells. Nevertheless, the path of signal transduction between the crucial contact of the cells with endotoxin (lipopolysaccharide) and the synthesis and release of the mediators is yet poorly understood. In particular, the involvement of Ca2+ and protein kinase C in this process is still a matter of controversy. Here, it will be demonstrated that removal of extracellular Ca2+ by EGTA does not have a significant effect on the endotoxin-stimulated production of tumor necrosis factor-alpha (TNF-alpha) and on total protein synthesis in rat Kupffer cells. However, the release of prostaglandin E2 could not be raised above the basal level under these conditions. Treatment with inhibitors of protein kinase C such as the isoquinoline derivative, H-7, or staurosporin is without influence on TNF-alpha synthesis. The depletion of protein kinase C through preincubation of rat Kupffer cells with phorbol 12-myristate 13-acetate for 24 h was also without effect on TNF-alpha production. The effectiveness of these inhibitors under the conditions used was ascertained by measurement of the O2- release from the same cell batches. Superoxide production known as protein kinase C-dependent in Kupffer cells (Dieter et al. (1986) Eur. J. Biochem. 86, 451-457) was suppressed in a dose-dependent manner by staurosporin or after prolonged pretreatment with the phorbol ester. H-7 decreased superoxide production only slightly in high doses that severely harm the Kupffer cells. Prostaglandin E2 release, although clearly protein-kinase C-dependent in phagocytosing rat Kupffer cells, is not decreased following exposure to lipopolysaccharide in the presence of protein kinase C inhibitors.