ADAMTS-like 2 (ADAMTSL2) is a secreted glycoprotein that is widely expressed during mouse embryogenesis and is regulated during skeletal myogenesis.

ADAMTS-like 2 (ADAMTSL2), is a secreted protein resembling the ancillary domains of the ADAMTS proteases, but with distinct structural features. It has 7 thrombospondin type-1 repeats (TSRs), but an unusually long spacer module, which in both humans and mice, contains a novel insertion bearing six N-glycosylation sites. The ADAMTSL2 protein expressed in HEK293F and COS-1 cells, is a cell-surface and extracellular matrix binding glycoprotein, with N-linked carbohydrate constituting approximately 20% by mass. The 4.0 kb Adamtsl2 mRNA is found most abundantly in adult mouse liver, lung and spleen by northern blotting. During mouse embryogenesis, Adamtsl2 was expressed most strongly in the third week of gestation. Adamtsl2 mRNA was detected by in situ hybridization in developing skeletal muscle, liver, bronchial and arterial smooth muscle, skin, intervertebral disc, perichondrium, pancreas and spinal cord. Immunohistochemical localization of ADAMTSL2 protein was similar to mRNA expression. Detection of Adamtsl2 mRNA and protein in developing skeletal myotubes, but not undifferentiated myogenic precursors led us to investigate its regulation during in vitro myogenic differentiation. In C2C12 and 23A2 myogenic cells, but not in 23A2 cells rendered non-myogenic by expression of G12V:H-Ras (9A2 cells), differentiation induced by serum starvation triggered expression of Adamtsl2 mRNA, coordinately with Myog, a marker of muscle differentiation. Furthermore, activation of the key myogenic determinant MyoD in 10T1/2 fibroblasts also triggered expression of Adamtsl2 mRNA. Collectively, the data suggest that induction of Adamtsl2 mRNA is an integral feature of myogenesis.     

Antimicrobial Activity of Propolis on Oral Microorganisms

Formation of dental caries is caused by the colonization and accumulation of oral microorganisms and extracellular polysaccharides that are synthesized from sucrose by glucosyltransferase of Streptococcus mutans. The production of glucosyltransferase from oral microorganisms was attempted, and it was found that Streptococcus mutans produced highest activity of the enzyme. Ethanolic extracts of propolis (EEP) were examined whether EEP inhibit the enzyme activity and growth of the bacteria or not. All EEP from various regions in Brazil inhibited both glucosyltransferase activity and growth of S. mutans, but one of the propolis from Rio Grande do Sul (RS2) demonstrated the highest inhibition of the enzyme activity and growth of the bacteria. It was also found that propolis (RS2) contained the highest concentrations of pinocembrin and galangin. Received: 8 June 1997 / Accepted: 7 July 1997     

Gradient-thickness thin-layer chromatography for the isolation and analysis of trace amounts of free fatty acids in large lipid samples

A thin-layer chromatographic method for quantitative isolation of free fatty acids is described. This method appears to be more satisfactory than existing methods in offering the combination of advantages of specificity, simplicity, rapidity, reproducibility, accuracy, high sensitivity, and applicability as a preparative technique. The method involves chromatography on a thin-layer plate on which the layer of Silica Gel G decreases linearly in thickness from 1000 micro at the base to 125 micro at the upper end. This gradient-thickness design allows the separation and densitometric quantitation of very small traces of free fatty acids from relatively large and complex lipid samples in a single chromatographic step. The method has been shown to be applicable directly to the crude total lipid extracts of several mammalian tissues. It appears to generate little if any artifactual free fatty acids from the breakdown of complex lipids, in contrast to the undesirable behavior of silicic acid columns in this respect. Gradient-thickness thin-layer chromatography promises to be useful for the quantitative isolation of trace amounts not only of other types of lipids but also of classes of compounds other than lipids.     

Stability of the brain fatty acid pattern in adult rats during extreme starvation

Abstract— Adult rats were denied food for 7 days. As compared with a control group, this severe starvation reduced the mean total body weight by 44 per cent, the weight of the diaphragm by 33 per cent and liver by 67 per cent, the total lipid content of the diaphragm by 57 per cent and liver by 69 per cent, and the total lipid phosphorus content of the diaphragm by 19 per cent and liver by 68 per cent. The decrease in lipid phosphorus contents indicates that the diaphragm and liver catabolized membrane phospholipids as well as triglycerides. In contrast, the fresh weight of the brain and the total lipid content of the brain were not significantly altered by starvation. The fatty acid patterns of the total lipid of the diaphragm and liver (determined by GLC) were grossly altered by starvation. In the brain, however, 17 of the 21 fatty acids measured did not change significantly (P > 0.05) and the remaining four changed, with borderline statistical significance, by only 2 to 13 per cent. There was no significant effect of starvation of the pattern of the brain polyunsaturated fatty acids when measured by alkali isomerization. In contrast to the liver and diaphragm, the brain is apparently unable to utilize its lipids appreciably as an energy source. Presumably the lipids of the brain are preserved to permit this organ to function properly, even in the last stages of starvation.     

Localization and distribution of Microccus lysodeikticus membrane ATPase determined by ferritin labeling

Antiserum to Ca²⁺-activated ATP phosphohydrolase (EC 3.6.1.3) isolated and purified from membranes of Micrococcus lysodeikicus was prepared in rabbits and guinea pigs. The γ-globulin fractions of these antisera reacted with and inhibited ATPase activity in isolated membranes but failed to absorb to intact protoplasts or purified mesosome fractions. ATPase activity was not detectable in the purified mesosomal preparations and trypsin treatment and sonication failed to release any activity. Ferritin conjugated to the γ-globulin fractions of the antiserum reacted with the ATPase particles on the membrane as visualized in negatively stained preparations examined in the electron microscope. Labeled membranes showed a distribution of ferritin very similar to the patterns observed for ATPase particles on untreated membranes. No significant labeling occurred when the ferritin conjugate was reacted with intact protoplasts or mesosome fractions. Thin sections of ferritin-labeled membranes established the asymmetric disposition of the ATPase, with the conjugate visible on only one side of the membrane. The results indicate that the ATPase protein occurs on the inner face of the membrane. All labeling experiments were verified immunologically. When ferritin-labeled membranes were subjected to the selective release procedure used in releasing the ATPase-like particles from the membranes, a complex of ferritin-conjugate associated with the ATPase particles was released. The selective release of ferritin-antibody-enzyme complexes from the membrane opens up a new way of studying the molecular architecture of cell membranes.     

Role of the Sympathetic Innervation of the Pacinian Corpuscle

An investigation was made into the nature of the role played by the noradrenergic innervation of the pacinian corpuscle. Corpuscles of the cat mesentery and mesocolon were used in all experiments. Blockade of noradrenergic beta receptors by dichloroisoproterenol and interference with norepinephrine release by reserpine are each capable of reversibly blocking mechanoelectric transduction by the pacinian corpuscle. The monoamine oxidase inhibitors iproniazid and phenelzine are capable of protecting the transducer from the blocking effects of reserpine. It is concluded that the presence of norepinephrine, as maintained by sympathetic tonus, is required for the afferent nerve terminal of the pacinian corpuscle to be mechanosensitive.     

Lethal effect of rifampicin in Bacillus subtilis as a complicating factor in the assessment of the lifetime of messenger ribonucleic acid

The bactericidal action of rifampicin was compared with that of chloramphenicol in growing and in sporulating cultures of Bacillus subtilis 168. Chloramphenicol kills cells only very slowly, but exposure to rifampicin kills over 95% of cells in a few minutes, causing gross physical damage, which is visible in both phase-contrast and electron microscopy. This is accompanied by a fall in O(2) consumption and by lysis. Experiments with synchronized cultures showed that susceptibility to the lethal effect of rifampicin is greater when the cells are dividing. The results suggest that the synthesis of some species of RNA other than mRNA may be necessary for the maintenance of cell integrity, although experiments with actinomycin D do not altogether fit this interpretation. However, we conclude that rifampicin is too toxic to use as an antibiotic for assessing the lifetime of mRNA.