Genetically modified Lactococcus lactis producing a green fluorescent protein-bovine lactoferrin fusion protein suppresses proinflammatory cytokine expression in lipopolysaccharide-stimulated RAW 264.7 cells.

Lactoferrin (LF), an iron-binding glycoprotein distributed widely in the biological fluids of mammals, is believed to play an important role in host defenses against infection. Previous studies in animal models and humans demonstrated that combined administration of LF and probiotic lactic acid bacteria (LAB) can prevent sepsis. In this study, we genetically engineered a probiotic LAB strain, Lactococcus lactis, to produce recombinant bovine LF based on the green fluorescent protein (GFP)-fused expression system. Western blotting confirmed that the genetically modified L. lactis strain (designated NZ-GFP-bLF) produced a protein corresponding to a fusion of GFP and bLF in the presence of nisin, an inducer of target gene expression. The protein synthesized by NZ-GFP-bLF was fluorescent and thus we monitored the time-dependent change in the production level of the recombinant protein using fluorometric analysis. The utility of NZ-GFP-bLF in preventing sepsis was determined by investigating its anti-inflammatory property in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Pretreatment of RAW 264.7 cells with NZ-GFP-bLF significantly attenuated the LPS-induced mRNA expression and protein production of 3 proinflammatory cytokines (IL-1α, IL-6, and tumor necrosis factor-α) compared with pretreatment with a vector control strain of L. lactis. Our results suggest that NZ-GFP-bLF holds promise for the development of a new prophylaxis for sepsis.

Immunostimulatory oligodeoxynucleotide containing TTTCGTTT motif from Lactobacillus rhamnosus GG DNA potentially suppresses OVA-specific IgE production in mice.

In a previous study of the immunoregulatory properties of commensal bacterial DNA, we identified the strong immunostimulatory oligodeoxynucleotide (ISS-ODN) ID35 in the genomic DNA of Lactobacillus rhamnosus GG (LGG). The observed effects of ID35 are because of the unique TTTCGTTT motif located at the 5' end of the ODN, which is different from the previously identified ISS motifs in humans and mice. In the present study, we used an ovalbumin (OVA)-sensitized mouse model to show that ID35 is a potent suppressor of antigen-specific immunoglobulin E (IgE) production in vivo. This effect was toll-like receptor 9-dependent, as GpC negID35 failed to suppress antigen-specific IgE production. ID35 activated the specific subset of CD11c+CD8a+ dendritic cells, which are associated with T-helper 1 (Th1)-type systemic responses, and effectively induced interferon-gamma (IFN-gamma) production by CD4+ T cells in OVA-challenged mice. These immunoregulatory effects of ID35 were comparable with those induced by the murine prototype ODN 1826. Thus, ID35 is the first ISS-ODN with such a strong immunostimulatory and IgE suppressor activity to be found in immunobiotic bacterial DNA.

delta 4-3-Oxosteroid 5 beta-reductase. Structure and function.

delta 4-3-Oxosteroid 5 beta-reductase catalysing reduction of delta 4-3-oxosteroids to give A/B cis-conformation was intraperitoneally injected into BALB/c strain mice with Ribi adjuvant. Monoclonal antibody specific for this enzyme was prepared from their spleen cells. Using this monoclonal antibody as a probe the enzyme was further purified using reversed phase liquid chromatography to determine amino-acid sequence protein-chemically. Attempts to determine the N-terminal amino acid failed, indicating that the N-terminal amino acid is blocked. The protein was therefore subjected to digestion with lysyl endopeptidase after alkylating with iodoacetate. The peptides thus formed were isolated and purified by reversed-phase high-performance liquid chromatography and their amino-acid sequences were determined. Using antibodies and oligonucleotides as probes a cDNA which contained a 978 bp long open reading frame encoding 326 amino-acid residues (Mr 37376) was isolated from rat liver cDNA libraries and the entire sequence of the protein was deciphered from its nucleotide sequence. The COS cells transfected with this cDNA revealed a versatile activity to reduce varied kinds of delta 4-3-oxosteroids, i.e. 7 alpha-hydroxy-4-cholesten-3-one, androstenedione and cortisone as postulated by Okuda and Okuda (1984, J. Biol. Chem. 259, 7519-7524) and Furuebisu et al. (1987, Biochim. Biophys. Acta 912, 110-114. With a newly established immunoblotting assay method several tissues and organs were surveyed and it was found that the enzyme exists only in the liver and there is an apparent difference between sexes as to the content of this enzyme. However, there was little if any difference in the amount of mRNAs between both sexes, which may indicates that the sexual difference of rat liver cytosol 5 beta-reductase is due to a posttranslational modification and/or degradation.

Molecular cloning and sequence analysis of cDNA encoding delta 4-3-ketosteroid 5 beta-reductase of rat liver.

A cDNA clone encoding delta 4-3-ketosteroid 5 beta-reductase was isolated from rat liver cDNA libraries using antibodies specific for the enzyme and oligonucleotides as probes. The cDNA contained 981-base pair open reading frame encoding 327 amino acid residues (Mr 37,376) and an unusually long 3'-untranslated region rich in AT sequence in the total length of 3189 base pairs. The predicted amino acid sequence contains the sequences similar to the putative NADPH- and steroid-binding regions.

A multifocal schwannoma of the masseteric nerve causing hemicrania.

A case is reported of schwannoma of the right masseteric nerve, invading the pterygopalatine fossa and the parapharyngeal space through the mandibular notch in a 61-year-old woman. The tumor in this case was composed of four various-sized interconnected nodules, simulating a string of beads. The tumor was the cause of the patient's chronic hemicrania, as confirmed by the relief from and disappearance of head pain after surgical removal of the tumor. The most important aspects of this case are the anatomic site of the schwannoma and its uncommon clinicopathologic manifestations; this tumor originated from the masseteric nerve and is the first case reported to straddle the mandibular notch.

Alterations of oncogenes in metastatic tumours of human gastric carcinomas.

To determine whether alterations in oncogenes are associated with tumour progression and metastasis, DNAs from 32 metastatic tumour samples of different sites in 12 autopsy cases of gastric carcinomas were analysed for alterations of ERBB, ERBB2, HST1, INT2 and LMYC genes by Southern blot hybridisation. DNAs from 89 primary gastric carcinomas including 69 advanced carcinomas and 20 early carcinomas were also examined. In primary tumours, no amplification was detected in early carcinomas, while amplification of ERBB and ERBB2 genes was detected in one (1.4%) and four (5.8%) out of 69 advanced carcinomas, respectively. In metastatic tumours, amplification of ERBB gene was detected in three metastatic tumours (9.4%), and all of them had allelic deletion of the LMYC gene. Regardless of histological type, amplification of ERBB2 gene was detected in 8 metastatic tumours (25.0%), out of which three tumours had coamplification of HST1 and INT2 genes. The incidence of ERBB2 amplification in metastatic tumours was significantly higher than that in primary tumours. These results indicate that multi-alterations in oncogenes might occur during tumour progression and metastasis of human gastric carcinomas.

The identification of a rabbit-transmitted cervical toxoplasmosis mimicking malignant lymphoma.

A case is presented of acquired cervical toxoplasmosis occurring in a 43-year-old male, which clinically mimicked malignant lymphoma. The histopathology of this case was probable toxoplasmic lymphadenitis. Serologic tests and the use of FITC-labeled antibodies revealed high levels of specific IgG antibodies in the serum and toxoplasmic antigens in paraffin sections of the patient, respectively. During survey of the infection route, it was learned that the patient's pet rabbit and three other rabbits of the same family line had cervicofacial lumps. The pet rabbit had high levels of toxoplasmic antibodies. Immunofluorescence tests on the infraorbital lump also revealed Toxoplasma gondii. Therefore, it was concluded that in this case the rabbit had transmitted Toxoplasma to the patient. The authors know of no other reports of toxoplasmosis transmitted by or through rabbit to human.

Multiple congenital epulides in a newborn: with special reference to reported cases in Japan.

This report describes a case of multiple epulides which simultaneously occurred on the maxilla and the mandible of a 6-day-old female baby. The clinical findings, diagnostic procedure, surgical treatment, histopathology, and 2-year follow-up of this case are presented. Based on the clinicostatistical and histopathologic investigations with special reference to the pertinent Japanese literature, a possible histogenetic etiology of this entity is discussed. Any odontogenic component might play an important role in the development of this tumor. This case of multiple epulides is considered to be very rare.

Phenotypic expression of human epidermal growth factor in foetal submandibular gland and pleomorphic adenoma of salivary gland.

The phenotypic expression of the human epidermal growth factor (EGF) was investigated immunohistochemically in human foetal submandibular glands from the 5th to 10th month of gestation, adult normal submandibular glands and 48 cases of pleomorphic adenomas. In foetal submandibular glands, both the terminal buds and primary ducts at the intermediate stage of gestation were positive for EGF, and in particular, the outer layer cells of primary ducts showed strong EGF-immunoreactivity. EGF-positive cells decreased as the gestational stage advanced and only ductal cells were weakly positive for EGF at the terminal stage of gestation. In the adult normal submandibular gland, weak immunoreactivity for EGF was restricted to ductal cells. However, 41 (86%) of the 48 pleomorphic adenomas had EGF-positive cells which were distributed among the ductal, chondroid and myxoid portion. No EGF-immunoreactivity was detected in the solid portion of pleomorphic adenomas. These results suggest that EGF may play an important role in the growth and differentiation of foetal cells as well as the proliferation of tumour cells in pleomorphic adenomas.