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.     

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.     

The insecticidal activity of some norditerpene dilactones

The effects on housefly (Musca domestica) of six norditerpene lactones (nagilactones B, D, E, podolide, hallactone B and 14-epi-ponalactone A) in a defined diet were tested. Nagilactone D was the most active, with an LD₅₀ of 0.7 ppm. Nagilactones C and D were also toxic to light-brown apple moth (Epiphyas postvittana) and codling moth (Laspeyresia pomonella).The relationship between lactone structure and toxicity to housefly is discussed.

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Zusammenfassung Viele aus Podocarpus-Arten isolierte Norditerpenlactone sind für Stubenfliegen toxisch. Es wird über die Resultate der Fütterung von sechs Lactonen an die Stubenfliege in einem definierten Nährboden berichtet und die LD₅₀ von zwölf Verbindungen werden verglichen. Der aktivste Stoff ist Nagilacton mit einer LD₅₀ von 0,7 ppm. Nagilacton C und D sind auch toxisch für Epiphyas postvittana und für den Apfelwickler. Die wirksamsten Verbindungen haben eine kurze nichtpolare Seitenkette und eine elektronenreiche, funktionelle Gruppe bei C-8, ferner sind sie Epoxyalkohole in Ring-A. Diese in der Natur vorkommenden Lactone spielen wahrscheinlich eine Schutzrolle in der Pflanze.

     

Distribution and movement of toxaphene in anaerobic saline marsh soils

The biological oxygen demand (BOD) of filtered water from Lake Wingra, Wisconsin is significantly higher in the littoral zone than in the pelagial zone. Laboratory experiments indicate that BOD is not influenced by water temperature at the time of sampling or by enrichment with nitrate or ammonia. Rather, enrichment with macrophyte leachate sharply increases BOD, and enrichment with phosphate produces a small but significant increase in BOD. We conclude that high BOD in littoral waters of the lake is an indication of production of labile organic matter in the water by dense beds of the macrophyte Myriophyllum spicatum.     

Multiple toxin production by an isolate of Aspergillus flavus

Three toxins were recovered from rice and wheat cultures of an isolate of Aspergillus flavus. The toxins were present simultaneously in the cultures after one or two weeks incubation and were identified as aflatoxin, cyclopiazonic acid and aflatrem, a recently identified indole-mevalonate metabolite.No endorsements are implied herein.     

Coppr deficiency in the rat. Relationship to chronic cyanide poisoning.

Daily administration of increasing doses intraperitoneally of 2.5-4.0 mg NaCN/kg to male Wistar rats for 5 weeks produced acute signs of poisoning immediately post-injection but no sign of chronic toxicity except lower final body weights than in control rats. CN-treated rats had less liver copper than controls, but not below the range of normality, and their liver mitochondrial membranes were 24% less able to bind adenine nucleotides than control membranes. No other biochemical or pathological sign of copper deficiency occurred. Liver cytochrome oxidase activity was normal after the 5 weeks of CN-administration, as was the ability of liver mitochondria to synthesize phospholipids. The ultrastructure of hepatocytes was normal without evidence of the enlarged, misshapen mitochondria produced by copper deficiency. Normal cytochrome oxidase activity of liver mitochondria, together with reduced liver copper levels and reduced binding affinity of mitochondrial membranes for adenine nucleotides, indicate that the membrane binding site for adenine nucleotides is not cytochrome oxidase per se but may involve copper, perhaps by virtue of its cationicity. With repeated exposure to CN- rats develop tolerance to acute poisoning. It is suggested that this may be due to the switch in glucose catabolism towards the pentose pathway at the expense of other pathways.