Establishment of an Animal Model Using Recombinant NOD.B10.D2 Mice To Study Initial Adhesion of Oral Streptococci

An oral biofilm is a community of surface-attached microorganisms that coats the oral cavity, including the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases in patients with dry mouth or Sjögren''s syndrome (SS). The purpose of this study was to establish an animal model for studying the initial adhesion of oral streptococci that cause biofilm formation in patients with dry mouth and SS in an attempt to decrease the influence of cariogenic organisms and their substrates. In nonobese diabetogenic (NOD) mice that spontaneously develop insulin-dependent diabetes mellitus (IDDM) and SS, we replaced major histocompatibility complex (MHC) class II (A^g7 E^g7) and class I D^b with MHC class II (A^d E^d) and class I D^d from nondiabetic B10.D2 mice to produce an animal model that inhibited IDDM without affecting SS. The adhesion of oral streptococci, including Streptococcus mutans, onto tooth surfaces was then investigated and quantified in homologous recombinant N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice. We found that a higher number of oral streptococci adhered to the tooth surfaces of N5 (NOD.B10.D2) and N9 (NOD.B10.D2) mice than to those of the control C57BL/6 and B10.D2 mice. On the basis of our observation, we concluded that these mouse models might be useful as animal models of dry mouth and SS for in vivo biological studies of oral biofilm formation on the tooth surfaces.Oral streptococci are present in large numbers in dental plaque, and several types interact with the enamel salivary pellicle to form a biofilm on tooth surfaces (9, 16, 17, 21, 29). Streptococci account for approximately 20% of the total number of salivary bacteria (24), with Streptococcus salivarius being the primary organism. Further, the densities of Streptococcus mutans and Streptococcus sanguis in saliva are more than 1 × 10⁵ cells per ml. S. mutans is a pioneering organism that plays an important role in biofilm formation on tooth surfaces and is a primary causative agent of dental caries (9, 16, 21). The mechanical forces of salivary flow and tongue movement tend to dislodge and expel bacteria from tooth surfaces and the oral cavity (3, 5, 6), and their importance in controlling microbial colonization in the oral cavity has been well demonstrated in individuals with diabetes mellitus, Sjögren''s syndrome (SS), and dry mouth, who suffer from a rapid overgrowth of biofilm and rampant caries, making them highly susceptible to oral infections (1-2, 6). Thus, attempts to investigate the initial adhesion by oral streptococci, including S. mutans, in mouse models are likely to aid in the understanding and prevention of oral infectious diseases caused by the components of oral biofilm.Previous studies of S. mutans infections in the oral cavities of mice have been performed by feeding the animals diets containing sucrose in the presence of glucans (13, 15, 30, 43). Since the adherence of S. mutans to the tooth surface may depend on the balance between physical adherence and synthesis of insoluble glucans in a natural environment, that infection method may be inappropriate for investigation of natural biofilm formation associated with streptococci, including S. mutans (18, 39).The nonobese diabetogenic (NOD) mouse strain is currently the best available model for the study of insulin-dependent type 1 diabetes mellitus (IDDM) and SS (11, 31), both of which develop spontaneously and are characterized by lymphatic infiltration of the pancreas and salivary glands. Oral changes are prominent features of these diseases, which are manifested by dry mouth and hyposalivation (6, 7, 37). NOD mice are also used as an animal model for the study of oral infectious diseases associated with systemic diseases such as diabetes and SS or dry mouth.The unique major histocompatibility complex (MHC) class II genes (I-A^g7, no expression of I-E) represent dominant susceptibility factors and mediate activated T cells during the development of diabetes in NOD mice (11, 22, 25, 36, 41, 42). In the NOD model of SS, histopathological analyses of the salivary glands in MHC-congenic strains of NOD mice have indicated that the I-A^g7 region is not required for lymphocytic infiltration (26, 31). Further, replacement of the NOD MHC class I K^d region with another haplotype, MHC class I K^wm7, as well as replacement of the MHC class II A^g7 E^g7 and class I D^d regions with the corresponding region from the other MHC haplotype, has been shown to prevent diabetes (12). However, replacement with MHC class I K does not completely prevent development of insulitis. In another report, NOD mice pretreated nasally by using peptides restricted with MHC class I K^d showed a delayed onset of spontaneous IDDM, though insulitis could not be prevented by the induction of tolerance (23).In the present study, we attempted to establish an animal model for oral infectious diseases such as dental caries by focusing on replacement of the MHC class II and class I D region but not the class I K region in nondiabetic NOD mice by outcrossing B10.D2 mice (K^d, I-A^d, and D^d) with NOD mice (K^d, I-A^g7, and D^b) because the MHC class I K region in B10.D2 mice is identical with that in NOD mice (12). The present backcrossed and intercrossed NOD mice with the MHC class II and MHC class I D region replaced with that from B10.D2 mice developed SS, however, not diabetes. We then attempted to determine whether these mice would be useful as animal models for a sucrose-free study of the initial adhesion of oral streptococci on tooth surfaces in humans.     

Pioglitazone improves the phenotype and molecular defects of a targeted Pkd1 mutant

Mutations of either PKD1 or PKD2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The molecular function of the gene product of PKD1, polycystin-1, in vitro has been elucidated recently, but the molecular pathological consequences of the loss of polycystin-1 in vivo have remained unclear. We have generated a mouse with a targeted deletion of exons 2-6 of Pkd1 to study the molecular defects in Pkd1 mutants. Homozygote embryos (Pkd1(-/-)) developed hydrops, cardiac conotruncal defects and renal cystogenesis. Total protein levels of beta-catenin in heart and kidney and c-MYC in heart were decreased in Pkd1(-/-) embryos. In the kidneys of Pkd1(-/-), the expression of E-cadherin and PECAM in basolateral membranes of renal tubules was attenuated, and tyrosine phosphorylation of epidermal growth factor receptor and Gab1 were constitutively enhanced when cystogenesis started on embryonic day (E) 15.5-16.5. Maternally administered pioglitazone, a thiazolidinedione compound, resolved these molecular defects of Pkd1(-/-). Treatment with pioglitazone improved survival of Pkd1(-/-) embryos and ameliorated the cardiac defects and the degree of renal cystogenesis. Long-term treatment with pioglitazone improved the endothelial function of adult Pkd1(+/-). These data indicated that molecular defects observed in Pkd1(-/-) embryos contributed to the pathogenesis of ADPKD and that thiazolidinediones had a compensatory effect on the pathway affected by the loss of polycystin-1. Pathways activated by thiazolidinediones may provide new therapeutic targets in ADPKD.     

Intracerebroventricular injection of brain natriuretic peptide inhibits vasopressin secretion in conscious rats

The effect of intracerebroventricular (i.c.v.) injection of brain natriuretic peptide (BNP), a novel peptide purified from the porcine brain, on arginine vasopressin (AVP) secretion was studied in conscious, unrestrained rats and was compared with that of atrial natriuretic polypeptide (ANP). I.c.v. administration of BNP (0.01, 0.1 or 1 nmol) significantly inhibited basal AVP secretion and the effect of BNP was comparable to that of ANP. The AVP secretion induced by i.c.v. injection of angiotensin II (0.1 nmol) was significantly suppressed by the pretreatment with BNP (0.1 or 1 nmol). These results suggest that BNP is involved in the central control of AVP secretion either alone or in combination with brain ANP.     

The effect of acetylcholine on chloride transport across the mouse lacrimal gland acinar cell membranes

The mechanisms of Cl^– transport and the effects of acetylcholine (ACh) and electrochemical Cl^– potential changes across the basolateral plasma membrane on intracellular Cl^– activity in the acinar cells of isolated mouse lacrimal glands were studied using double-barreled Cl^–-selective microelectrodes. In the resting state, the basolateral membrane potential (V _m) was about –40 mV and intracellular Cl^– activity was about 35 mmol/l. Addition of ACh (10^–910^–6 mol/l) hyperpolarizedV _m and decreased the Cl^– activity in a dose-dependent manner. ACh (10^–6 mol/l) hyperpolarizedV _m by 20 mV and decreased the cytosolic Cl^– activity with an initial rate of 16.0 mmol/l · min. Reduction of the perfusate Cl^– concentration to 1/9 control depolarizedV _m and decreased cytosolic Cl^– activity at a rate of 1.9 mmol/l · min. AV _m hyperpolarization of 20 mV produced by DC injection to the adjacent cell decreased Cl^– activity at a rate of 4.6 mmol/l · min. DIDS (1 mmol/l) hyperpolarizedV _m by 8 mV with little change in Cl^– activity and increased the input resistance of the cells by 25%. DIDS decreased the rate of change in Cl^– activity induced by low-Cl^– Ringer to 35% of control, but had no effect on the ACh-evoked decrease in the Cl^– activity. Furosemide (1 mmol/l) slightly hyperpolarizedV _m and decreased Cl^– activity at a slow rate but affected Cl^– movements induced by ACh or low-Cl^– Ringer only slightly. Cl^– uptake into the cells was inhibited partially by furosemide. The present results showed that ACh induces an increase in the Cl^– permeability across the luminal plasma membrane and that the basolateral membrane possesses a DIDS-sensitive Cl^– conductance pathway and a furosemide-sensitive Cl^– uptake mechanism.     

No association of GSK3beta gene (GSK3B) with Japanese schizophrenia.

Several lines of evidence indicate that glycogen synthase kinase-3beta (GSK3beta) is one of the candidates for schizophrenia-susceptibility factor. However, it has not been reported the association analysis between GSK3beta gene (GSK3B) and Japanese schizophrenia based on linkage disequilibrium (LD). We provide an association analysis using relatively large samples (381 schizophrenia, and 352 controls) after determination of "tag single nucleotide polymorphisms (SNPs)." In this LD mapping, we selected and genotyped for eight polymorphisms (seven SNPs and one diallelic (CAA)(n) repeat), which covered the entire region of GSK3B, and determined two "tag SNPs." In the following association analysis using these two "tag SNPs," we could not find association with Japanese schizophrenia. Furthermore, we also include subgroup analysis considering age-at-onset and subtypes, neither could we find associations. Because our samples provided quite high power, these results indicate that GSK3B may not play a major role in Japanese schizophrenia.     

The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization

Choroidal neovascularization (CNV) is directly related to visual loss in some eye diseases, such as age-related macular degeneration. Although several human histological studies have suggested the participation of macrophages in CNV formation, the precise mechanisms are still not fully understood. In this study, we elucidated the role of ocular-infiltrating macrophages in experimental CNV using CCR2 knockout (KO) mice, wild-type mice, and C57BL/6 (B6) mice. CCR2 is the receptor of monocyte chemoattractant protein-1, and the number of infiltrating macrophage and the area of CNV were significantly reduced in CCR2 KO mice. Enriched ocular-infiltrating macrophages from B6 mice actually showed angiogenic ability in a dorsal air sac assay. Moreover, their expression of class II, CD40, B7-1 and B7-2 molecules, and the mRNA for potential angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor alpha, was also observed. Collectively, we conclude that ocular-infiltrating macrophages play an important role in CNV generation.     

Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T-T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) alpha and beta, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER alpha, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER alpha-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER alpha may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.     

Effects of various compounds on histidine decarboxylase activity: Active site mapping

The effect of about one hundred compounds on the activity of histidine decarboxylase partially purified from whole bodies of fetal rats was determined. Most of them at their 10 mM concentration had little effect on the enzyme activity; but 12 compounds inhibited the enzyme to a greater extent than 30%. Among these, except for -methylhistidine that has been known to be a strong and specific inhibitor, DOPA, homocysteine, cysteine, methionine and urocanic acid were the best inhibitors; -phenyllactic acid, phenylpyruvic acid and carnosine were less strong inhibitors; valine, oxaloacetic acid andN -methylimidazole acetic acid were weak inhibitors. Histamine had no inhibitory action. Thus, the substrate binding site of histidine decarboxylase is very rigid and specific forl-histidine.