Role of nitric oxide in murine cytomegalovirus (MCMV) infection

Cytomegalovirus (CMV) is a typical pathogen of an opportunistic infection. In this review article, various roles of nitric oxide (NO) in murine CMV (MCMV) infections, including acute, persistent and latent infections, are discussed. In the acute phase of MCMV infection, NO plays a protective role against MCMV infection. In contrast, NO has been proven to act as a pathogenic factor in a model of MCMV pneumonitis. In MCMV persistent infection, when MCMV was detected only in the salivary gland, T cells of mice were modified to produce a massive amount of such cytokines as TNF-alpha and IFN-gamma upon in vivo stimulation with anti-CD3. These cytokines then induced mRNA for inducible NO synthase (iNOS), thus resulting in the production of a large amount of NO. A histochemical study demonstrated that NO damaged bronchial epithelial cells, and thereby apparently inducing pneumonitis. In the case of a latent infection, when viral DNA was detected in the host in spite of the absence of any infectious particle, NO increased the amount of persistently-infected MCMV-DNA. As a result, NO was found to act as "a double edged sword" in the CMV-host relationship.     

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.     

NEDD8 recruits E2-ubiquitin to SCF E3 ligase

NEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modifier that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8-modifying system accelerates the formation of the E2-E3 complex, which stimulates protein polyubiquitylation.     

alpha-Adrenoceptor stimulation-mediated negative inotropism and enhanced Na(+)/Ca(2+) exchange in mouse ventricle

Mechanisms underlying the negative inotropic response to alpha-adrenoceptor stimulation in adult mouse ventricular myocardium were studied. In isolated ventricular tissue, phenylephrine (PE), in the presence of propranolol, decreased contractile force by approximately 40% of basal value. The negative inotropic response was similarly observed under low extracellular Ca(2+) concentration ([Ca(2+)](o)) conditions but was significantly smaller under high-[Ca(2+)](o) conditions and was not observed under low-[Na(+)](o) conditions. The negative inotropic response was not affected by nicardipine, ryanodine, ouabain, or dimethylamiloride (DMA), inhibitors of L-type Ca(2+) channel, Ca(2+) release channel, Na(+)-K(+) pump, or Na(+)/H(+) exchanger, respectively. KB-R7943, an inhibitor of Na(+)/Ca(2+) exchanger, suppressed the negative inotropic response mediated by PE. PE reduced the magnitude of postrest contractions. PE caused a decrease in duration of the late plateau phase of action potential and a slight increase in resting membrane potential; time courses of these effects were similar to that of the negative inotropic effect. In whole cell voltage-clamped myocytes, PE increased the L-type Ca(2+) and Na(+)/Ca(2+) exchanger currents but had no effect on the inwardly rectifying K(+), transient outward K(+), or Na(+)-K(+)-pump currents. These results suggest that the sustained negative inotropic response to alpha-adrenoceptor stimulation of adult mouse ventricular myocardium is mediated by enhancement of Ca(2+) efflux through the Na(+)/Ca(2+) exchanger.     

Nkx2.5 and Nkx2.6, homologs of Drosophila tinman, are required for development of the pharynx

Nkx2.5 and Nkx2.6 are murine homologs of Drosophila tinman. Their genes are expressed in the ventral region of the pharynx at early stages of embryogenesis. However, no abnormalities in the pharynges of embryos with mutations in either Nkx2.5 or Nkx2.6 have been reported. To examine the function of Nkx2.5 and Nkx2.6 in the formation of the pharynx, we generated and analyzed Nkx2.5 and Nkx2.6 double-mutant mice. Interestingly, in the double-mutant embryos, the pharynx did not form properly. Pharyngeal endodermal cells were largely missing, and the mutant pharynx was markedly dilated. Moreover, we observed enhanced apoptosis and reduced proliferation in pharyngeal endodermal cells of the double-mutant embryos. These results demonstrated a critical role of the NK-2 homeobox genes in the differentiation, proliferation, and survival of pharyngeal endodermal cells. Furthermore, the development of the atrium was less advanced in the double-mutant embryos, indicating that these two genes are essential for both pharyngeal and cardiac development.     

New compound heterozygous mutation in the CYP17 gene in a 46,XY girl with 17 alpha-hydroxylase/17,20-lyase deficiency

BACKGROUND: 17alpha-Hydroxylase/17,20-lyase deficiency is caused by a defect of P450c17 which catalyzes both 17alpha-hydroxylase and 17,20-lyase reactions in adrenal glands and gonads. RESULTS: In the present study, we analyzed the CYP17 gene in a Japanese patient with 17alpha-hydroxylase/17,20-lyase deficiency. The patient was a phenotypic girl and referred to us for right-sided inguinal hernia at the age of 4 years. Biopsy of the herniated gonad showed testicular tissue. The karyotype was 46,XY. At 6 years of age, hypertension was clearly recognized and the patient was diagnosed as having 17alpha-hydroxylase/17,20-lyase deficiency based on the clinical and laboratory findings. Analysis of the CYP17 gene revealed a compound heterozygous mutation. One mutation was an undescribed single nucleotide deletion at codon 247 in exon 4 (CTT to CT: 247delT) and the other was a missense mutation resulting in a substitution of His to Leu at codon 373 in exon 6 (CAC to CTC: H373L), which has been previously shown to abolish both 17alpha-hydroxylase and 17,20-lyase activities. The functional expression study of the 247delT mutant showed that this 247delT mutation completely eliminates both 17alpha-hydroxylase and 17,20-lyase activities. CONCLUSIONS: Together, these results indicate that the patient is a compound heterozygote for the mutation of the CYP17 gene (247delT and H373L) and that these mutations inactivate both 17alpha-hydroxylase and 17,20-lyase activities and give rise to clinically manifest 17alpha-hydroxylase/17,20-lyase deficiency.     

Enhanced Tolerance Against Salt-Stress and Freezing-Stress of Escherichia coli Cells Expressing Algal bbc1 Gene

The expression of the eukaryotic bbc1 (breast basic conserved) gene (the bbc1 gene of the marine green alga Chlamydomonas sp. W-80 strain) enhanced the tolerance against salt-stress and freezing-stress in E. coli cells. The expression of the BBC1 protein in the E. coli cells carrying the algal bbc1 gene and that in the Chlamydomonas W-80 cells were examined by Western blotting analysis. The result suggests that the eukaryotic BBC1 protein expressed in the E. coli cells has a protective function against the cellular dehydration. Received: 24 April 2000 / Accepted: 21 August 2000     

Construction of humanized anti-ganglioside monoclonal antibodies with potent immune effector functions

 Gangliosides GD3, GD2 and GM2, which are the major gangliosides expressed on most human cancers of neuroectodermal and epithelial origin, have been focused on as effective targets for passive immunotherapy with monoclonal antibodies. We previously developed a chimeric anti-GD3 mAb, KM871, and a humanized anti-GM2 mAb, KM8969, which specifically bound to the respective antigen with high affinity and showed potent immune effector functions. Humanization of anti-ganglioside antibody is expected to enhance its use for human cancer therapy. In the present study, we generated a chimeric anti-GD2 mAb, KM1138, and further developed the humanized form of anti-GD2 and anti-GD3 mAbs by the complementarity-determining regions grafting method. The resultant humanized anti-GD2 mAb, KM8138, and anti-GD3 mAb, KM8871, showed binding affinity and specificity similar to those of their chimeric counterparts. In addition, both humanized mAbs had functional potency comparable to the chimeric mAbs in mediating the immune effector functions, consisting of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The production of these humanized anti-ganglioside mAbs, with potent effector functions and low immunogenicity, precedes the evaluation of the therapeutic value of anti-ganglioside mAbs in passive immunotherapy and the target validation for ganglioside-based vaccine therapy. Received: 30 November 2000 / Accepted: 30 January 2001     

Desulfurization of Benzothiophene by the Gram-Negative Bacterium, Sinorhizobium sp. KT55

Sinorhizobium sp. KT55 was the first Gram-negative isolate to be capable of utilizing benzothiophene as the sole source of sulfur. By GC-MS analysis of metabolites of benzothiophene by this strain, benzothiophene sulfone, benzo[e][1,2]oxathiin S-oxide and o-hydroxystyrene were detected, suggesting that the benzothiophene desulfurization pathway of this strain is benzothiophene → benzothiophene sulfoxide → benzothiophene sulfone → benzo[e][1,2]oxathiin S-oxide →o-hydroxystyrene. Desulfurization activity of this strain was significantly repressed by methionine, cysteine, sulfate, dimethyl sulfoxide, and Casamino acids. Received: 5 January 2001/Accepted: 6 February 2001