Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture

Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with d-[1-³H]glucosamine and sodium [³⁵S]sulphate. Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet. Sulphated glycosaminoglycans co-chromatographed when eluted by NaCL gradient from DEAE-cellulose. The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate. Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid. Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units. Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain. The N:O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1. Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of $\text{‘O’-}\text{sulphate}$ groups differed strikingly between heparan sulphates from the medium and cell-surface compartments. A di-O-sulphated tetrasaccharide was identified in both heparan sulphate species. The absence of detectable amounts of ³⁵[S]sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups.     

Rapid intracellular degradation of newly synthesized collagen by bone cells. Effect of dichloromethylenebisphosphonate

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesized collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [3H]proline into collagenase-digestible proteins or by the conversion of [3H]proline into [3H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.     

Developmental Expression of HNK-1-Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.     

Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.     

Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase.

It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.     

Localisation of degradative enzymes in white-rot decay of lignocellulose

The use of immunogold-cytochemical labelling techniques in electron microscopy of wood infected by basidiomycete fungi has assisted in the elucidation of the localisation of enzymes which degrade lignocellulose. The use of specific immunocytochemical techniques is discussed with respect to the authenticity and accuracy of the methods, the use of adequate controls in the gold-labelling procedure, and the immunospecificity of the antibodies.Localisation of the lignin-degrading enzymes, lignin-peroxidase and laccase, has shown that these enzymes do not bind to wood cell walls unless the process of decay has already commenced. Similarly localisation of cellulases Endoglucanase II (EGII) and Cellobiohydrolase I (CBHI) has shown that these enzymes only bind to exposed ends of cellulose fibrils and to partially degraded areas of the wood cell wall. -Glucosidase is always immobilised within the extracellular polysaccharide layer surrounding fungal hyphae.This review postulates that there is regulation of the release sequence of these lignocellulolytic enzymes defining the spatial arrangement between the hyphae and the wood cell wall. This hypothesis is presented diagrammatically.     

The synthesis of proteoglycans by human T lymphocytes

We have examined the proteoglycans produced by highly-purified cultures of human T-lymphocytes. The proteoglycans were metabolically labelled with [35S]sulphate and analysed in cellular and medium fractions using DEAE-cellulose chromatography, gel filtration and specific enzymatic and chemical degradations. The results showed that the T cells synthesized a relatively homogeneous, proteinase-resistant chondroitin 4-sulphate proteoglycan that accumulated in the culture medium during a 48 h incubation period. The cellular fraction contained a significant amount of free chondroitin sulphate chains that were not secreted into the medium. These polysaccharides were formed by intracellular degradation of proteoglycan in a chloroquine-sensitive process, indicating a requirement for an acidic environment. In contrast to chondroitin sulphate derived from proteoglycan, chondroitin sulphates synthesized on the exogenous primer, beta-D-xyloside, were mainly secreted by the cells. beta-D-Xylosides caused an 8-fold stimulation in the synthesis of chondroitin sulphate, but decreased the synthesis of proteoglycan by about 50%. These proteoglycans contained shorter chondroitin sulphate chains than their normal counterparts. The results indicate that although proteoglycans are mainly secretory components in human T-cell cultures, a specific metabolic step leads to the intracellular accumulation of free glycosaminoglycans. Separate functions are likely to be associated with the intracellular and secretory pools of chondroitin sulphate.     

Evidence for distinct polygenic regulation of antibody responses to some unrelated antigens in lines of mice selected for high or low antibody responses to somatic antigen of Salmonella

The effect of the selective breeding of mice for high or low antibody production to complex immunogens is largely nonspecific, since it modifies the responsiveness of high (H) and low (L) lines to many antigens unrelated to the selection antigen. However, the nonspecific effect of the polygenic control operating in these lines is not a general feature. For example, the group of genes in selection IV, carried out for responsiveness to somatic antigen of Salmonella, does not modify the responses to sheep erythrocytes (SE). Despite equivalent responses in H and L mice of selection IV, a large variability was found in individual responses of F₂ interline hybrids, which demonstrates the presence of alleles with high or low effect on responses to SE. A selective breeding (Selection IV-A) was therefore initiated from this F₂ population for responsiveness to SE. A progressive interline divergence occurred during the first seven generations of selection; the interline separation was due to polygenic regulation (about four independent loci from a preliminary estimate).Equivalent responses to the s antigen of Salmonella are observed in the two lines. This constitutes additional evidence for distinct polygenic regulation of responses to SE and to somatic antigen. Moreover, the pattern of responses to several unrelated antigens (nonspecific effect) also differs between Selections IV and IV-A.Abbreviations H high responder lines - L low responder lines - s somatic antigen of Salmonella - f flagellar antigen of Salmonella - R response to selection - S selection differential - F₀ foundation population - h² heritability (realized) - RGG rabbit gamma globulin - CE chicken erythrocyte - HE human erythrocyte - PE pigeon erythrocyte - SE sheep erythrocyte     

Synaptonemal complexes with modified lateral elements in Sordaria humana: development of and relationship to the “recombination nodules”

Meiotic prophase in Sordaria humana has been analyzed by three-dimensional reconstructions of 3 leptotene, 2 zygotene, 10 pachytene and 3 diplotene nuclei. Several notable features emerged. The lateral components of the synaptonemal complexes (SC) are hollow tubes which show dilations of variable sizes from late leptotene to early diplotene. These bulges occur before pairing. Their number decreases as soon as the SC are completely formed, but their mean size increases. Bulges can be present in all parts of the lateral components including telomeres and nucleolar organizer region, but their distribution along bivalents is not random. The remarkably uniform width of the SC central region, normally observed in other species is not observed in S. humana. Although as a general rule the bulges rarely affect the homologous components at identical sites, they often either fill or partially cover the central region. The recombination nodules are not clearly connected with the bulges. This work provides also additional insight into the development of both SC and the nodules. At late leptotene, the homologues are aligned before SC formation. One case of interlocking has been observed at early pachytene. Nodules are present from zygotene to diplotene. They are not evenly distributed along the bivalents during pachytene. The mean number of nodules, constant from late pachytene to diplotene, is equal to the mean number of chiasmata.