Differential expression of decorin and biglycan genes during palatogenesis in normal and retinoic acid-treated mice.

Proteoglycans are involved in secondary palate formation. In the present study, we focused on two small leucine-rich proteoglycans, decorin and biglycan, because they assembled extracellular matrix molecules such as collagens and modulated signaling pathway of transforming growth factor-beta. To investigate the functions of decorin and biglycan in palatogenesis, we compared their mRNA expression patterns between normal palate and retinoic acid-induced cleft palate in mice by using in situ hybridization analysis during the period of embryonic day 13.5 (E13.5) to E15.5. On E13.5, decorin mRNA was expressed in the epithelia and mesenchyme on the nasal side of the developing secondary palate. During the period the palate shelves were fusing (E14.5), decorin mRNA was strongly expressed in the mesenchyme but its expression pattern was asymmetric; decorin mRNA expression area in the nasal side was broader than that in the oral side. The expression of decorin mRNA was hardly detected in the mesenchyme on either side of the medial edge epithelium. After fusion (E15.5), its expression converged to the mesenchyme just around the palatine bone. Biglycan mRNA was ubiquitously distributed throughout the palatal mesenchyme for the mid-gestation period. Its expression area became limited to the ossification area within the palate after the late gestation period. In the retinoic acid-treated mice, the area of the decorin gene expression expanded to the core region of the palate primordium where little signal was observed in control mice. On the other hand, biglycan in the retinoic acid-treated mice did not show remarkable change in its distribution patterns compared with that in the control mice. These findings suggest that decorin and biglycan play distinct roles in palatogenesis, and decorin was more actively involved in the process of secondary palate formation than biglycan. Up-regulation of decorin gene expression in the retinoic acid-treated mice might influence the pathogenesis of cleft palate.     

A study of the action of picrotoxin on the inhibitory neuromuscular junction of the crayfish

1. The effect of picrotoxin on the neuromuscular junction of the crayfish (Cambarus clarkii) was investigated. The potential changes were recorded intracellularly and extracellularly with micro-electrodes. The membrane conductance of the muscle fibre was also measured.2. Picrotoxin depressed the amplitudes of the inhibitory junctional potentials and the potential changes produced by iontophoretically applied gamma-aminobutyric acid (GABA), but had no appreciable effect on the excitatory junctional potentials and the potential changes produced by L-glutamate.3. The presynaptic action of GABA and the neural transmitter was depressed by picrotoxin. The presynaptic action of beta-guanidinopropionic acid was also depressed by picrotoxin.4. The increase in the membrane conductance produced by the addition of GABA in the bath fluid was depressed by picrotoxin. The dose-response relation showed that picrotoxin depressed the conductance increase produced by GABA in a non-competitive manner. The action of picrotoxin on the conductance increase produced by GABA was more effective in low Cl- solution.5. The analysis of the dose-response curves showed that the action of picrotoxin was well expressed by the Michaelis-Menten equation, but the slope of the dose-response curve of GABA was steeper than this relation. It is proposed that the conductance of the junctional membrane was increased by the combination of two molecules of GABA with a receptor, and the attachment of one molecule of picrotoxin to a specific site depressed the conductance increase.     

New genome type of adenovirus serotype 19 causing nosocomial infections of epidemic keratoconjunctivitis in Japan

Twelve strains of adenovirus serotype 19, isolated from cases of epidemic keratoconjunctivitis in Japan in 1992, 1993, 1997, and 1998, were analyzed by DNA restriction analysis, using restriction endonucleases BamHI, BglI, BglII, EcoRI, HindIII, KpnI, PstI, SacI, SalI, SmaI, and XhoI. Among these 11 restriction endonucleases, EcoRI, PstI, SacI, and SmaI were discriminative enzymes, showing restriction patterns different from those reported previously for the prototype and the variant 19a. This new genome type was isolated in 1997 and 1998, when an increase of epidemic keratoconjunctivitis cases caused by adenovirus serotype 19 was observed for both sporadic and nosocomial infections. Strains from 1992 and 1993 showed restriction patterns similar to those of the worldwide reported variant 19a for all enzymes used. The changes detected in strains from 1997 and 1998 could be the reason for the recent epidemic.     

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani

Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66 – 100% for isolates of different subgroups within an AG, and 55 – 96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance. Received: 11 February /16 May 1997     

Developmental expression of carbonic anhydrase-related proteins VIII,X, and XI in the human brain

Three cDNA homologues of carbonic anhydrase with unknown biological functions have been reported: carbonic anhydrase-related proteins (CA-RP) VIII, X, and XI. In the present study, we produced monoclonal antibodies to these CA-RPs and studied their regional and cellular distributions in the human adult and fetal brains by immunohistochemical analysis. In the adult brain, CA-RP VIII was expressed in the neural cell body spreading to most parts of the brain. CA-RP X was expressed in the myelin sheath and its expression was shown in the cytoplasm of cultured tumor cells by immunocytochemical analysis. CA-RP XI was expressed in the neural cell body, neurites, and astrocytes in relatively limited regions of the brain. In the fetal brain, CA-RP VIII and XI were expressed in the neuroprogenitor cells in the subventricular zone as early as the 84th day of gestation and subsequently detected in the neural cells migrating to the cortex. CA-RP X first appeared in the neural cells in the cortex at the 141st day. In the choroid plexus, the epithelial cells gave CA-RP VIII and XI expressions in both adult and fetal brains.From the findings in the present study on the distribution and the developmental expression of CA-RP VIII, X, and XI in the human brain we suggest that these CA-RPs play roles in various biological process of the CNS.     

Renal sodium transport abnormality: Gitelman's syndrome and renal sodium transporter

Recent studies using molecular biological methods have enabled us to identify the genetic abnormality in renal electrolyte metabolism. In renal tubules, diuretic sensitive Na transporter systems are present, and key molecules have been cloned. Thiazide-sensitive Na-Cl contransporter (TSC) is one of the molecules localized in the distal convoluted tubule, whose genetic abnormality causes Gitelman's syndrome (a variant of Bartter's syndrome characterized by dehydration, hypokalemic metabolic alkalosis, secondary aldosteronism lacking hypertension, hypomagnesemia, and hypocalciuria). We identified a mutation in TSC (Leu to Pro change at 623 amino acid position, L623P) in familial Gitelman's syndrome, and we confirmed the loss of TSC function by this mutation in a functional expression system using mammalian cells. This L623P mutation has been found in other patients with Gitelman's syndrome living in the northern part of Japan.     

Mucosal gastrin receptor. V. Development in newborn rats

We determined the development of the oxyntic gland mucosal gastrin receptor in rats killed at various times from 5 to 60 days after birth. Rats were weaned on the 18th day after birth. Newborn animals had no detectable gastrin binding, high serum gastrin levels (800-1,200 pg/ml), low antral gastrin levels (0.5-2.0 micrograms/g tissue), or high pH of gastric contents (pH greater than 5.0) and did not respond to pentagastrin. At the time of weaning, serum gastrin dropped to 600 pg/ml and reached adult levels (300 pg/ml) on day 40. Antral gastrin increased to 7.5 micrograms/g tissue on day 20 and reached adult levels (20 micrograms/g tissue) on day 22. Specific binding of gastrin was first detected on day 20 and reached the adult level of 4 fmol/mg protein on day 60. Pentagastrin significantly stimulated acid secretion on day 20 and DNA synthesis on day 25. Prevention of weaning through day 25 decreased the magnitude but did not prevent or delay the onset of the above changes. These results indicate that 1) the absence of a gastrin response in newborn rats is due to a lack of gastrin receptors, 2) development of gastrin receptor and biological sensitivity to gastrin appear at the time of weaning, and 3) the development that occurs with weaning is enhanced but not triggered by the shift to solid food.