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.     

TFIIA induces conformational changes in TFIID via interactions with the basic repeat.

DNA-binding studies with Saccharomyces cerevisiae TFIID point mutants indicated that TFIIA interacts with the basic repeat region of TFIID and induces structural changes. The latter was shown by the ability of TFIIA to compensate for TFIID point mutants defective for DNA binding. Interaction with TFIIA also rendered TFIID binding temperature independent, thus mimicking the effect of removing the nonconserved N terminus of TFIID. In addition, N-terminal truncation of the TFIID point mutants defective for DNA binding mimicked the ability of TFIIA to restore DNA binding of those mutants. Taken together, these results suggest that TFIIA enhances TFIID binding to DNA by eliminating an otherwise inhibitory effect of the nonconserved N terminus of TFIID. Furthermore, analyses of TFIID contact points on DNA and binding studies with TATA-containing oligonucleotide probes showed that TFIIA decreases the effect of sequences flanking the adenovirus major late TATA element on TFIID binding to DNA, suggesting a possible role of TFIIA in allowing TFIID to recognize a wider variety of promoters.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov.

Based on the 16S rRNA sequences, DNA-DNA homology values, cellular lipid and fatty acid composition, and phenotypic characteristics, a new genus Burkholderia is proposed for the RNA homology group II of genus Pseudomonas. Seven species in this group were transferred to the new genus. Thus seven new combinations, Burkholderia cepacia (Palleroni and Holmes 1981), Burkholderia mallei (Zopf 1885), Burkholderia pseudomallei (Whitmore 1913), Burkholderia caryophylli (Burkholder 1942), Burkholderia gladioli (Severini 1913), Burkholderia pickettii (Ralston et al 1973) and Burkholderia solanacearum (Smith 1896) were proposed.     

Identification of a CA repeat at the TCRA locus using yeast artificial chromosomes: a general method for generating highly polymorphic markers at chosen loci.

The creation of a comprehensive genetic map in human has been limited by the lack of highly polymorphic markers spaced evenly throughout the human genome. We have utilized yeast artificial chromosomes (YAC) containing large human DNA inserts to help identify highly polymorphic (CA)n repeats at a chosen locus. The DNA of a YAC containing the locus was subcloned in M13 vectors, and the recombinants were screened at high stringency to detect preferentially long (CA)n repeats (n greater than 20). These repeats, which are the most likely to be highly polymorphic, were then studied to confirm both the level of polymorphism and their precise genetic location. This strategy has permitted the identification of a new, highly polymorphic CA repeat (77% heterozygosity) at the T cell receptor alpha chain (TCRA) locus on chromosome 14q. It provides a powerful marker for assessing the role of this locus in the susceptibility to autoimmune and infectious diseases. This approach should permit the development of highly polymorphic markers at any targeted locus and rapidly improve the current human genetic map.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

Increase in serotonin 5-HT1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia.

Binding studies with [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), a specific serotonin1A (5-HT1A) receptor agonist, were done on the autopsied brains from control subjects and from patients with chronic schizophrenia. All the patients and controls were of the Japanese race. In the controls, representative Scatchard plots for the specific [3H]8-OH-DPAT bindings in the prefrontal cortex and hippocampus revealed a single component of high affinity binding site (Kd value = 5.7 and 5.9 nM, Bmax value = 80.1 and 101.0 fmol/mg protein, respectively). The [3H]8-OH-DPAT bindings to the prefrontal cortex and hippocampus were potently inhibited by serotonin (IC50 = 6.3 x 10(-9) M) and 5-HT1A agonists (IC50 = 5.0 x 10(-9) - 2.3 x 10(-7) M), while other neurotransmitters, 5-HT2 and 5-HT3 related compounds did not inhibit the binding (IC50 greater than 10(-5) M). The bindings were decreased in the presence of 0.1mM GTP and 0.1mM GppNHp but not in the presence of 0.1mM GMP. In the prefrontal and temporal cortices of schizophrenics, there was a significant increase in the specific [3H]8-OH-DPAT binding, by 40% and 60%, respectively, with no change in the hippocampus, amygdala, cingulum, motor cortex, parietal or occipital cortex, as compared to findings in the controls. Scatchard analysis showed that this increased binding reflects changes in the number of sites but not in the affinity. The effect of 0.1mM GppNHp on the binding to prefrontal cortex was observed in both controls and schizophrenic patients. The bindings were significantly greater in the schizophrenic patients than in controls, in the presence of 0.1mM GppNHp. Our findings suggest that there are GTP-sensitive 5-HT1A sites in the human brain and that selective increases in GTP-sensitive 5-HT1A sites in the prefrontal and temporal cortices of schizophrenics relate to the pathophysiology of schizophrenia.     

TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells.

The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells.     

Changes of carp myosin subfragment-1 induced by temperature acclimation

Summary Heavy meromyosin subfragment-1 (S1) was prepared by -chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca²⁺- and K⁺ (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 mol P_i·min^-1·mg^-1, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (K_D) of S1 from cold-acclimated carp was 32.1x10^-4· s^-1, compared to 13.2x10^-4·s^-1 for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg²⁺-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s^-1 with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - K _m apparent dissociation constant - P _i inorganic -phosphate - PMSF phenylmethane-sulfonyl fluoride - S ₁ heavy meromyosin subfragment-1 - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TPCK N-tosyl-l-phenylalanyl chloromethyl ketone - V _max maximum initial velocity