Single-locus control of the mast cell population in mouse skin

The number of mast cells in connective tissue from dorsal skin varied markedly among mouse strains. Inbred strains of mice were typed into three groups, high (NC and NZB mice), low (B6, B10, and BALB/c mice), and intermediate (C3H/He and DBA/2 mice), by their mast cell content in the skin. However, the strain differences in the number of mast cells was marginal at the age of weaning but became distinct with age. This could be explained mainly by the frequently observed clustering of mast cells in adult NC and NZB mice and the rarely observed clustering in younger mice as well as in adult B10 and BALB/c mice. The breeding experiment revealed that the difference in the number of mast cells between NC and B10 mice was controlled by a single autosomal dominant locus, for which we propose the designation Mcr (mast cell regulator). The role of the Mcr locus with regard to the frequency of the mast cell population in connective tissue is discussed.     

Enzymatic reactions in aqueous-organic media. IV. Chitin-alpha-chymotrypsin complex as a catalyst for amino acid esterification and peptide synthesis in organic solvents

Summary Chitin--chymotrypsin complex was found to be an effective catalyst for the esterification of N-acetyl-L-tryptophan in ethanol and for peptide synthesis from N-acetyl-L-tyrosine and glycinamide in acetonitrile. In the former reaction both initial rate of ester formation and equilibrium yield of the ester markedly increased by the complexation of chymotrypsin with chitin compared to free chymotrypsin.     

dam methylation and the initiation of DNA replication on oriC plasmids

Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.     

Lipoamidase (lipoyl-X hydrolase) from pig brain.

Although the optimum substrate for lipoamidase (lipoyl-X hydrolase) has not yet been determined, it is known that lipoamidase activity, as determined by hydrolysis of the synthetic substrate lipoyl 4-aminobenzoate (LPAB), is widely distributed in pig brain tissues, i.e. in the cerebrum, cerebellum and medulla. Over 95% of the enzyme activity is present in the membrane subfractions, indicating that brain lipoamidase is an integral membrane protein enzyme. To elucidate the chemical nature and the optimum substrate of the abundant lipoamidase in the brain, we isolated it from the membrane subfractions. After an 8-step purification procedure, brain lipoamidase was purified 601-fold and identified as a 140 kDa glycoprotein by SDS/PAGE. A mechanistic study to determine Km and Vmax, values was carried out using various synthetic compounds. Lipoyl-lysine, which is generally believed to be a naturally occurring substrate of lipoamidase, was first compared with biotinyl-lysine, because these two vitamins have reactive sulphur atoms and are similar in molecular mass and structure. The Km for lipoyl-lysine was 333 microM, whereas biotinyl-lysine was not hydrolysed. Stringent specificity for the lipoyl moiety is demonstrated, as expected. Dipeptides of amino acid-lysine structures were studied, and dipeptides of aspartyl- and glutamyl-lysine hydrolysis occurred at high Km (3 mM) values. Thus lysine in the moiety is not very effective as an optimum substrate. The chemical bond structures of the amide bond (lipoyl-lysine) and peptide bond (aspartyl-lysine) were hydrolysed. Next, the ester bond compound was tested, and it was observed that lipolylmethyl ester was hydrolysed at high specificity. These findings indicate that this enzyme has broad specificities with respect to bond structure; it therefore is a unique hydrolase having stringent specificity for lipoic acid and relatively broad specificity for the chemical bond and the X moiety. Various inhibitors were tested; a few reagents, such as organic mercurials, di-isopropylfluorophosphate, 1,10-phenanthroline, sodium azide and angiotensin-converting enzyme inhibitor exhibited some inhibition (not more than 60%). Thus the active centre of this enzyme is a complex type. Although ATP is not hydrolysed and the lowest Km value is exhibited by the synthetic substrate reduced from LPAB (12 microM), some other compounds may still be expected to be hydrolysed by this unique and abundant brain lipoamidase.     

Microfibrils: a constitutive component of reticular fibers in the mouse lymph node

Summary Fibrous components other than collagen fibrils in the reticular fiber of mouse lymph node were studied by electron microscopy. Bundles of microfibrils not associated by elastin and single microfibrils dispersed among collagen fibrils were present. The diameter of the microfibrils was 13.29±2.43 nm (n=100). Elastin-associated microfibrils occurred at the periphery of the reticular fiber. Elastin was enclosed by microfibrils, thus forming the elastic fiber, which was clearly demonstrated by tannic acid-uranyl acetate staining. In the reticular fiber of lymph nodes, the elastic fiber consisted of many more microfibrils and a small amount of elastin. These microfibrils, together with the collagen fibrils, may contribut to the various functions of the reticular fibers.     

Accumulation of collagen III at the cleft points of developing mouse submandibular epithelium

The distribution of collagens I, III, IV and V was studied by immunoperoxidase staining of early developing mouse submandibular glands. Collagen I was always present in the extracellular matrices of the mesenchyme and at the epithelial-mesenchymal interfaces of the 12-day gland with no clefts and of the 13-day gland with a few definite clefts. Collagen III was found in a similar fashion to that of collagen I in the mesenchyme, but the distribution at the epithelial-mesenchymal interfaces was very different. In the mid 12-day gland with a round lobule, collagen III was distributed at every slightly indented site of basal epithelial surfaces. At the late 12-day stage, a few initial signs of cleft appeared on the surface, at which accumulation of collagen III became evident. Intense immunoreaction of collagen III in the early 13-day gland was seen at the bottom of every narrow cleft. No specific accumulation of collagens IV and V was observed in clefts of the late 12-day and early 13-day glands. Staining of collagen III in the 12-day gland cultured for 10 h in the presence of bovine dental pulp collagenase inhibitor, which has been shown to stimulate cleft initiation, was very prominent at the bottom of every narrow cleft. These observations suggest that collagen III works as a key substance for either in vitro or in vivo cleft initiation of the mouse embryonic submandibular epithelium.     

The role of interstitial collagens in cleft formation of mouse embryonic submandibular gland during initial branching

An interstitial collagenase was purified from the explant medium of bovine dental pulp and was shown to degrade collagens I and III but not IV and V. The enzyme halted cleft initiation in the epithelium of 12-day mouse embryonic submandibular glands in vitro, indicating the active involvement of interstitial collagens in the branching morphogenesis. Transmission electron microscopic observation of the intact 12-day gland without any clefts showed the scattered localization of a few collagen fibrils at the epithelial-mesenchymal interface of the bulb and also revealed the presence of numerous microfibrils around the stalk. Collagen bundles were regularly seen close to the wavy basal lamina at the bottom of clefts of the intact 13-day gland and 12-day gland cultured for 17 h under normal conditions. Mesenchymal cells were found in the clefts together with the frequent localization of peripheral nerve fibres and capillary endothelial cells. The collagen bundles were more often observed in the 12-day gland cultured in the presence of bovine dental pulp collagenase inhibitor, which had been shown to enhance cleft formation. In contrast, collagen fibrils were rarely found at the epithelial-mesenchymal interface of the 12-day gland cultured in the presence of Clostridial or bovine dental pulp collagenase. The findings indicated that the formation of interstitial collagen bundles is essential to form clefts in the epithelium both in vivo and in vitro.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.