Production and immunohistochemical characterization of a monoclonal antibody raised to proteoglycan purified from a human yolk sac tumour

Summary A large proteoglycan with chondroitin sulphate and dermatan sulphate side chains has been isolated and purified from a yolk sac tumour of the left ovary from a 23-year-old female. A monoclonal antibody, designated 2B1, was produced which reacted specifically with the intact molecule of the large proteoglycan and the chondroitinase ABC-treated core molecule. The localization of substances showing cross-reactivity to this antibody was studied in a variety of human tissues by means of indirect immunohistochemistry. The interstitial elements of nearly all tissues of a 5-month-old foetus were intensely reactive with the antibody, but in adult tissues structures that gave positive reactions were limited; only the perivascular and perimuscular fibrous elements were reactive, except for the aorta, which reacted extensively. In contrast, the interstitial elements of the carcinoma tissues tested were intensely reactive. Thus antibody 2B1 can be regarded as a useful tool for studies on the immunohistochemical localization of large proteoglycan in various human tissues.     

In vitro and in vivo recombination-related reactions of Escherichia coli recA protein and glucosyl-hydroxymethyl-deoxycytidine DNA

Summary Recombination of T4 phage is not controlled by the host recA gene but by an analogous phage gene, uvsX. We have tested the hypothesis that recA protein is inactive in T4-infected cells because it is unable to catalyze reactions involving single stranded DNA containing glucosyl-hydroxylmethyl-deoxycytidine. We found, however, that with modified and unmodified deoxycytidine containing DNAs, uvsX protein and recA protein catalyze in vitro reactions related to DNA recombination, but in T4-infected cells recA protein fails to promote strand transfer of DNA which contains unmodified deoxycytidine.Abbreviations dC-DNA deoxycytidine containing DNA - dC-T4 T4 phage containing dC-DNA - dHMC-DNA glucosyl-hydroxymethyl-deoxycytidine containing DNA - dsDNA double stranded DNA - gp gene product - ssDNA single stranded DNA     

Bovine Colostral Antibody Against Verotoxin 2 Derived from Escherichia coli O157:H7: Resistance to Proteases and Effects in Beagle Dogs

A bovine colostral antibody against verotoxin (VT) 2 of Escherichia coli O157:H7 was administered orally to beagle dogs. The antibody remained in the dogs’ small intestine for at least 2 h, whereas little serum antibody remained 1.5 h after administration. Furthermore, the antibody activity of secretory IgA did not change until 2 h after administration; however, the activity of IgG and IgM antibodies decreased by approximately 60% and 40% at 2 h after administration, respectively. Seven beagle dogs inoculated with Escherichia coli O157:H7 producing VT2 were administered bovine colostral antibody or bovine colostral whey without antibody. With administration of bovine colostral whey without antibody, the amount of VT2 in feces decreased gradually after administration and increased again at 5 d after inoculation, whereas bovine colostral antibody significantly reduced the amount of VT2 in feces on the day after administration. In addition, 9 beagle dogs were given bovine colostral antibody, bovine plasma antibody, or saline. The amount of VT2 in feces again decreased significantly more rapidly after administration of bovine colostral antibody than after administration of bovine plasma antibody or saline.Abbreviations: EHEC, enterohemorrhagic Escherichia coli; VT, verotoxinIn July 1996, a widespread outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection occurred among schoolchildren in Sakai, Japan, followed by numerous other similar outbreaks of food poisoning throughout the country.^4,19 Escherichia coli O157:H7 infection is monitored in Japan, in accordance with the Infection Diseases Control Law, and in 2005, 3589 cases were reported.¹⁰ Enterohemorrhagic Escherichia coli infection occurs in many industrialized nations²¹ and is an emergent infectious disease of significant clinical importance.^12,13,23Therapeutic approaches for EHEC infection are the subject of widespread discussion.^9,25,31 Generally, the treatment for bacterial food poisoning is antibiotic administration. However, antibiotic therapy is not recommended for food poisoning caused by EHEC infection, because it increases the risk of serious complications, such as hemolytic uremic syndrome, due to the release of verotoxin (VT) from killed bacteria. Therefore, alternative therapeutic approaches, such as inhibiting VT activity or absorption from the intestine, are required. We previously obtained a colostral antibody against VT2 from cows immunized with the toxin and confirmed the neutralization efficacy of this reagent against VT2 in mice.¹⁵ However, before this bovine colostral antibody can be administered to patients infected with E. coli O157, its resistance to decomposition by intestinal proteases must be investigated. Each immunoglobulin class reportedly differs in its resistance to protease degradation in vitro,^{1,3,18, 22,26,28} but such resistance has not been confirmed in vivo. Furthermore, few animal models are available for evaluating for E. coli O157:H7 infection. The weaned immature mouse model has been used to study E. coli O157:H7 infection and VT,¹⁵ and beagle dogs pretreated with fradiomycin before inoculation with E. coli O157:H7 developed diarrhea. We chose to use this canine model in the current study.In this study, we investigated the resistances of bovine colostral antibody and individual immunoglobulin classes to proteases in the small intestine of beagle dogs. We also evaluated the efficacy of this colostral antibody against VT2 in beagle dogs.     

Mutant HNF-1alpha and mutant HNF-1beta identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1alpha and mutant HNF-1beta in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1alpha and 13 mutant HNF-1alpha, as well as wild HNF-1beta and 2 mutant HNF-1beta, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1alpha and wild HNF-1beta significantly transactivated DPP-IV promoter, but mutant HNF-1alpha and mutant HNF-1beta exhibited low transactivation activity. Moreover, to study whether mutant HNF-1alpha and mutant HNF-1beta change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1alpha or wild HNF-1beta, or else respective dominant-negative mutant HNF-1alphaT539fsdelC or dominant-negative mutant HNF-1betaR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1alpha cells and wild HNF-1beta cells, whereas they decreased in HNF-1alphaT539fsdelC cells and HNF-1betaR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1alpha and wild HNF-1beta have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1alpha and mutant HNF-1beta attenuate the stimulatory effect.     

Localization of Type-2 Angiotensin II Receptor in Adrenal Gland

The localization of the type-2 angiotensin II receptor (AT2) in the adrenal glands of rats, guinea pigs, bovines, and humans was examined at the mRNA and protein levels. PCR products for AT2 were detected in the adrenal cortices and adrenal medullae of all the mammals examined with an RT-PCR technique. Three different anti-AT2 antibodies (Abs), whose specificity was confirmed in our hands, recognized a 50-kDa protein in the adrenal glands of the four mammals, and this recognition was abolished by the preabsorption of an Ab with an antigen. Immunoblotting and immunohistochemistry revealed that the 50-kDa protein was expressed consistently and variably in the adrenal cortices and medullae of various mammals, respectively. We conclude that the 50-kDa AT2 is consistently expressed in the adrenal cortex in a wide variety of mammals. (J Histochem Cytochem 58:585–593, 2010)     

Preparation of connexin43‐integrated giant Liposomes by a baculovirus expression–liposome fusion method

Connexin‐43 (Cx43) containing giant liposomes (GL) were prepared by a baculovirus expression–liposome fusion method. Recombinant budded viruses expressing Cx43 were prepared and then fused with GLs containing DOPG/DOPC at pH 4.5. Connexon formation on the GL membrane was observed by transmission electron microscope. Hydrophilic fluorescent dye transfers were observed through a Cx43‐mediated pathway not only between Sf9 (Spodoptera frugiperda) cells with Cx43 but also from giant Cx43 liposomes to Cx43‐expressing U2OS cells (human osteosarcoma cell). The functional connexin‐containing liposome is expected to be useful for cellular cytosolic delivery systems. The original orientation and function of Cx43 was maintained after integration into the liposomes. The liposome fusion method will create new opportunities as a tool for analysis of channel membrane proteins. Biotechnol. Bioeng. 2010;107: 836–843. © 2010 Wiley Periodicals, Inc.