Assimilatory reduction of nitrate in Rhizobium meliloti

Nitrate and nitrite reductases in the crude extract of aerobically grown Rhizobium meliloti were determined with methylviologen as electron donor at pH 7. Nitrate reductase was detected in the cells grown in the medium that did not contain nitrate, and in the presence of nitrate the specific activity increased about 2-fold. Nitrite reductase was induced by nitrate and produced ammonia from nitrite. In nitrate reducing cells, two kinds of O₂ labile nitrate reductase were found. One enzyme had optimal pH at 7 and was stabilized to O₂ by treating with DEAE-Toyopearl 650M. The other had optimal pH at 9 and was stabilized by the addition of dithiothreitol and EDTA. Nitrate reductase stabilized by DEAE-Toyopearl 650M treatment was purified 3,360-fold from crude extract. The purified enzyme showed a single protein band in polyacrylamide gel electrophoresis, and there was no absorption peak in the visible region. It had a molecular weight of 64,000 in SDS PAGE and 58,000 on Sephadex G-100 gel filtration. K_m for nitrate was 0.9 mM. It was inhibited by p-chloromercuribenzoate, cyanide, and α,α'-dipyridyl.     

Allergic conversion of protective mucosal immunity against nasal bacteria in patients with chronic rhinosinusitis with nasal polyposis

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Molecular interactions between presenilin and calpain: inhibition of m-calpain protease activity by presenilin-1, 2 and cleavage of presenilin-1 by m-, mu-calpain

Several mutations of presenilin (PS)-1, 2 result in early onset Alzheimer disease. Using the yeast two-hybrid system, the interaction between PS2 loop domain and the C-terminal region of mu-calpain was previously identified. Calpain is a calcium dependent-protease and there are two isoforms, m-calpain and mu-calpain, which differ in the calcium concentration required for activation. m-Calpain needs about 10(-3) M calcium ions, whereas mu-calpain about 10(-5) M. When PS and calpain were separately expressed in COS cells by cDNA transfection and then combined in vitro, or both were co-transfected to be co-expressed in vivo in COS cells, PS1 and PS2 reduced the casein proteolysis activity of m-calpain but not that of mu-calpain. Some of the PS mutations related to Alzheimer disease decreased this inhibitory activity. On the other hand, PS1 was cleaved by m-calpain and mu-calpain at a different site from those already reported (constitutive cleavage or alternative cleavage). These results suggest a regulatory function of presenilin on the calpain system.     

Expression of Fc receptors is suppressed in alveolar macrophages from patients with sarcoidosis.

To study the expression of Fc receptors in human alveolar macrophages (AM), the cells were collected from 12 healthy controls and 22 patients with sarcoidosis and the activity involved in binding to 125I-soluble immune complexes (IC) was investigated. The binding of 125I-IC was significantly suppressed in cells from the patients. A Scatchard plot analysis revealed that this suppression was due to a reduction in the number of Fc receptors on the surface membrane and not the result of a decrease in the binding affinity of the receptors. Rosette formation was observed by incubating AM with antibody-coated ox red blood cells. The presence of rosette forming cells was about 70% in the AM from healthy controls, while, a rate of only 50% was seen in those with sarcoidosis. These results indicate that the decrease of the 125I-IC binding in the AM from patients with sarcoidosis is to some extent the result of an increase in the population of the Fc receptor negative macrophages.     

HLA-Bw54 (Bw22-J, J-1) Antigen in Juvenile Onset Diabetes Mellitus in Japan

HLA—B8 and/or Bw15, associated with juvenile onset insulin-dependent diabetes mellitus (JDM) in Caucasians, have a very low frequency in the Japanese population. Thus, we were interested in investigating the association between JDM and HLA antigen in a Japanese population. Eighty-nine patients with JDM and 128 unrelated random controls were HLA-typed by the micro-lymphocytotoxicity test. The data revealed a significant, positive association between this type of diabetes mellitus and HLA-Bw54 (Bw22-J, J-1) antigen (Japanese-specific split antigen of HLA—Bw22).     

Reduction of enzymatic activity of tyrosine hydroxylase by a heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1), was due to reduced affinity to a cofactor biopterin.

A carcinogenic, food-derived heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1) was found to reduce the enzymatic activity of tyrosine hydroxylase in clonal rat pheochromocytoma PC12h cells, by its supplement to the culture medium. The reduction was observed with 10 microM Trp-P-1, and at this concentration the amount of cell protein and the activity of a non-specific enzyme, beta-galactosidase, were not affected. The mechanism of the reduction of the enzyme activity was clarified by kinetical studies. The amine reduced the affinity of tyrosine hydroxylase to a cofactor, tetrahydrobiopterin. The alteration of the enzymatic properties by Trp-P-1 was discussed in relation to the possible effect on catecholamine metabolism in the brain.     

Directed differentiation of telencephalic precursors from embryonic stem cells

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.     

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM. Received: August 30, 2001 / Accepted: November 2, 2001