Expression of connexin 32 gap junction protein in the kidneys during fetal development of the hamster (Mesocricetus auratus)

The expression of gap junction protein was examined immunohistochemically using affinity-purified antibody against rat liver gap junction protein, connexin 32 (Cx32), in the kidneys of fetal (gestation days 13–16) and adult Syrian golden hamsters. Phalloidin histochemical staining, PNA- and RCA I-lectin stainings, NCAM immunostaining, and alkaline phosphatase and Na⁺-K⁺-ATPase enzyme-histochemical staining were performed in combination with Cx32 immunostaining. The kidney sections were observed with a confocal scanning laser microscope. By gestation day 13, Cx32 immunoreactivity was observed in the differentiating tubules. The Cx32 staining was localized on the lateral cell membrane of the cells lining the developing proximal tubules, while the S-shaped bodies, developing distal tubules, and collecting tubules showed no positive immunostaining. As the kidney developed, the density of Cx32 immunoreactivity increased. As the gap junction provides pathways for cell-cell communication, the development of Cx32 expression may imply that this structure plays an important role in renal tubule development. Confocal scanning laser microscopy provided a clear image of the fluorescence-labeled cell structures, free from out-of-focus blur. Using the same sections, stereoscopic images were easily reconstructed from serial optical sections, and were helpful in understanding the spatial distribution of Cx32 expression in the developing fetal proximal tubules.     

HISTOPATHOLOGICAL STUDY ON PROGRESSIVE MUSCULAR DYSTROPHY—REPORT OF A CASE WITH AUTOPSY FINDINGS—

A typical case of the D uchenne type of progressive muscular dystrophy with autopsy findings was presented. Changes in the myocardial and smooth muscle of many organs were found, and the skeletal muscles also revealed florid changes.
Histopathological examination of the skeletal muscle was made in detail through light and electron microscopic observation.     

Immunohistochemical expression of MAM-3 and MAM-6 antigens in salivary gland tumours

Summary MAM-3 and MAM-6 antigens of human milk fat globule membrane were detected immunohistochemically in 93 cases of salivary gland tumours as well as in normal glands. The antigens were visualized in 10% formalin-fixed paraffin sections. MAM-3 (MoAbs 115G3, 67D11) antigen was distributed in intercalated and striated duct cells of the normal salivary glands, and in luminal tumour cells and squamous metaplastic cells of pleomorphic adenomas. In pleomorphic adenomas the frequency of positive staining with MoAb 67D11 (54/67; 80.6%) was higher than that with MoAb 115G3 (36/67; 53.7%). MAM-6 (MoAbs 115D8, 115F5) antigen was expressed in luminal and lateral borders of serous acinar cells and ductal of the normal glands, and also in luminal borders of tubulo-ductal and glandular structures of salivary gland tumours. Ductal basal cells were characterized by existence of positive staining for MAM-6 antigen, in adenolymphomas MAM-6 antigen was restricted to the basal tumour cells. Some mucous cells of mucoepidermoid tumours were stained specifically with MoAb 115G3, and epidermoid cells of mucoepidermoid carcinomas manifested MAM-6 antigen staining. Immunohistochemical localization of MAM-6 antigen resembled that of epithelial membrane antigen (EMA) detected with MoAb.     

Enhancement of Susceptibility to Shiga Toxin-Producing Escherichia coli O157:H7 by Protein Calorie Malnutrition in Mice

Infection with Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli is increasing among children. In this study, 5-week-old C57BL/6 mice with protein calorie malnutrition (PCM) that had been fed a 5% protein diet for 2 weeks since ablactation were inoculated intragastrically with 2 × 10⁶ CFU of Stx-producing E. coli O157:H7. More than 75% of infected mice with PCM died by 10 days postinfection. Infected mice with PCM developed neurologic symptoms 5 days after infection, while well-nourished control mice receiving a 25% protein diet did not. In the intestinal tracts of infected mice with PCM, inoculated E. coli O157:H7 multiplied between days 2 and 4 of infection, with a peak of growth at day 4. Although the pathogens were not culturable from the stool after day 7, O157 lipopolysaccharide was detectable in the stool by enzyme-linked immunosorbent assay even after day 8. Stx was detectable in the stool after day 2 of infection and increased in proportion to the growth of inoculated organisms. The maximal production of Stx occurred at 4 days postchallenge, and Stx was detectable in the blood on days 3 to 5. In contrast, well-nourished control mice survived the infection, and all of them remained well even after 3 weeks of infection. In these control mice, inoculated E. coli O157:H7 disappeared from the stool before day 3. Stx was not detectable in the stool and blood of infected control mice at any time from day 1 through day 8. Histologically, cerebral hemorrhages seemed to be the cause of acute death of infected mice with PCM. Immunocytochemical staining demonstrated the positive immunoreaction to Stx at the alveus and stratum pyramidale of the hippocampus and in renal tubules of infected malnourished mice. Such immunoreactions were not found in tissues from infected control mice. Histological study of the intestinal epithelium before infection showed that PCM severely affected the development of intestinal epithelia. These findings strongly indicate that PCM-induced nondevelopment of intestinal physical barrier is one of the predisposing factors for infection with Stx-producing E. coli O157:H7 in mice and suggest that our mouse model may explain the high incidence of infection with Stx-producing E. coli O157:H7 in the children whose intestinal epithelia have not yet completely developed.     

Immunocytochemistry of keratan sulfate proteoglycan and dermatan sulfate proteoglycan in porcine tooth-germ dentin

Keratan sulfate proteoglycan and dermatan sulfate proteoglycan have been reported to inhibit collagen fibrillogenesis. We investigated their distribution in order to evaluate the role of proteoglycan in dentinogenesis. Specimens of porcine tooth-germ dentin and erupted teeth were the materials on which antibodies to keratin sulfate and dermatan sulfate proteoglycan were used. Predentin was found to be positive for both antibodies and the reaction ceased in the calcification front. Uniformly thick collagen fibrils (30-70 nm in diameter) were distributed in the predentin matrix, which would become intertubular dentin in the future. Both antibodies reacted positively along these fibrils. In contrast, along the surface layer of dentin in the tooth germ and that in erupted teeth, collagen fibrils of 10-300 nm in diameter were noted occasionally in dentinal tubules whose odontoblastic processes had disappeared and these heterogeneous fibrils were negative for both antibodies. Our findings suggest that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed in the predentin inhibit calcification of collagen fibrils in the uncalcified matrix and disappear in the calcification front. It is further suggested that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed along collagen fibrils in the predentin matrix maintain uniform thickness, whereas collagen fibrils in dentinal tubules varied in thickness because of the absence of involvement of both proteoglycans. Therefore, keratan sulfate proteoglycan and dermatan sulfate proteoglycan were thought to be involved in both calcification and matrix formation.     

Enhancement of lipopolysaccharide-stimulated cyclooxygenase-2 mRNA expression and prostaglandin E2 production in gingival fibroblasts from individuals with Down syndrome

It is well known that Down syndrome (DS) is a premature ageing syndrome. Periodontal disease in individuals with DS develops rapidly and extensively in a relatively younger age bracket compared with that in healthy controls. The mechanisms involved in the periodontal inflammatory processes in DS patients are not fully understood. In the present study, the non-inflamed gingival fibroblasts isolated from seven patients with DS (DGF) and seven healthy controls (NDGF) were stimulated with lipopolysaccharide (LPS) derived from Actinobacillus actinomycetemcomitans (A. a.). We measured the level of prostaglandin E2 (PGE2) production by DGF and NDGF by radioimmunoassay, and also measured the mRNA expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) by using the real-time PCR method. We found the higher levels of LPS-stimulated COX-2 mRNA expression and PGE2 production in DGF when compared with those in NDGF. This study may indicate that overexpression of LPS-stimulated COX-2 induced a greater ability of DGF to produce PGE2, and that these phenomena may be responsible for the severer periodontal disease in DS patients.     

Streptococcus mutans Genes That Code for Extracellular Proteins in Escherichia coli K-12

Chromosomal DNA from Streptococcus mutans 6715 (serotype g) was cloned into Escherichia coli K-12 by using the cosmid pJC74 cloning vector and a bacteriophage λ in vitro packaging system. Rabbit antiserum against S. mutans extracellular proteins was used for immunological screening of the clone bank. Twenty-one clones produced weak to strong precipitin bands around the colonies, but only after the λ c1857 prophage was induced by being heated to lyse the E. coli cells. None of the clones expressed enzyme activity for several known S. mutans extracellular enzymes. One of these clones contained a 45-kilobase recombinant plasmid designated pYA721. An 8.5-kilobase fragment of S. mutans DNA from pYA721 was isolated and recloned into the BamHI restriction site of the plasmid vector pACYC184 to construct pYA726. pYA726 contained all, or nearly all, of the gene for a surface protein antigen (the spaA protein) of S. mutans 6715. This was deduced from immunological studies in which extracts of cells harboring pYA726 reacted with antisera against both purified 6715 spaA protein (about 210,000 daltons) and the immunologically similar antigen I/II of serotype c strains of S. mutans. In addition, the S. mutans spaA protein was found to possess at least one antigenic determinant not present on the protein specified by pYA726. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of E. coli clone extracts revealed that pYA726 produced a polypeptide with a molecular mass of about 180,000 daltons which was predominantly found in the periplasmic space of E. coli cells. Antisera to the spaA protein of S. mutans reacted with extracellular protein from representative strains of S. mutans serotypes a, c, d, e, f, and g, but not b.