Site-directed mutagenesis of stable adenosine triphosphate synthase

Evidence was obtained that four ionizable residues in the alpha and beta subunits of thermophilic ATP synthase (TF0F1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. TF0F1 was used because it is the only ATP synthase whose alpha-, beta- and gamma-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its beta-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting beta I164 and beta N252 subunits were both noncatalytic after re-assembly into the alpha beta gamma-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the alpha-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting alpha I175 subunit reassembled weakly into an oligomer, while the alpha N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an alpha beta gamma-complex that showed no inter-subunit cooperativity.     

A case of large prolactinoma supposed to be cured by bromocriptine therapy

The authors reported a patient with a large prolactinoma (PRL 1,716 ng/ml) who was treated with bromocriptine for two years and followed up for a subsequent 36 months. After the start of the therapy, the tumor size was dramatically reduced, and finally the disappearance of the tumor was confirmed by high resolution coronal CT. The serum prolactin level and pituitary function were normalized. The tumor has not regrown and the blood prolactin level has remained normal for 36 months since the discontinuation of bromocriptine administration. This is a very rare case report on the eradicative effect of bromocriptine on such a large prolactinoma. Another characteristic of this case was that the prolactin reserve was maintained not only before the therapy but also during the early stage of the therapy.     

A fusion gene in man: DNA sequence analysis of the abnormal globin gene of Hemoglobin Miyada

An abnormal globin gene from a patient heterozygous for Hemoglobin Miyada was cloned and sequenced. The results indicated that the 5′ flanking region and the 5′ side of the gene were identical to those of a β-globin gene and that the 3′ side was identical to that of a γ-globin gene. The part of the gene identical to a β-globin gene shifted to the part identical to the δ-globin gene somewhere in a homologous sequence region between the third nucleotide of the 17th codon and the second nucleotide of the 22nd codon of these two genes. Thus, results of analysis of the nucleotide sequence support the idea that the abnormal globin gene of Hemoglobin Miyada was generated as a fusion gene by unequal crossing over between a β- and a δ-globin gene.     

Defective gene in lactic acidosis: abnormal pyruvate dehydrogenase E1 alpha-subunit caused by a frame shift.

A patient with lactic acidosis showed a lowered pyruvate dehydrogenase E1 activity and fatigued on slight exercise. The cDNA encoding the pyruvate dehydrogenase E1 alpha-subunit from his lymphocytes, transformed by infection of Epstein-Barr virus, was cloned and sequenced. The nucleotide sequence determination revealed that the gene had a deletion of four nucleotides at the second codon upstream from the termination codon. This deletion would lead to a reading-frame shift and make a new termination codon at the 33d codon downstream from the "normal" termination codon. An S1 nuclease-protection experiment confirmed the presence of mRNA with its deletion in the patient. Amplification, by the polymerase chain reaction method, of the genomic-DNA region from his peripheral blood cells showed that the deletion was localized in an exon and that it was not caused by an abnormal splicing at the intron/exon junction. This is the first report on cloning a defective gene of the pyruvate dehydrogenase complex.     

Deficiencies in complex I subunits of the respiratory chain in Parkinson's disease

Immunoblotting studies on mitochondria prepared from the striata of patients who died of Parkinson's disease were performed using specific antisera against Complexes I, III and IV. In 4 out of 5 patients with Parkinson's disease, the 30-, 25- and 24-kDa subunits of Complex I were moderately to markedly decreased. No clear difference was noted in immunoblotting studies on subunits of Complexes III and IV between the control and Parkinson's disease. Deficiencies in Complex I subunits seem to be one of the most important clues to elucidate pathogenesis of Parkinson's disease.     

Multiple deletions in mitochondrial DNA at direct repeats of non-D-loop regions in cases of familial mitochondrial myopathy

Muscle mitochondrial DNAs from two brothers with mitochondrial myopathy associated with peripheral neuropathy had multiple deletions, most of which started in non-D-loop regions, unlike in an autosomal dominant mitochondrial myopathy (Zeviani, M. et al., Nature 339, 309 (1989)). The non-D-loop regions with deletions were amplified by the polymerase chain reaction and the resulting fragments were subcloned and then sequenced. At least 12 deletions of different lengths in different sites were found. However, all the deletions were flanked by short direct repeats (4-12 base pairs).     

Characterization of a cell-surface protein antigen of hydrophilic Streptococcus mutans strain GS-5

Fourteen strains of Streptococcus mutans serotype c were examined for their cell-surface protein antigens in terms of hydrophobicity, Mr and immunochemical specificities. Thirteen strains were hydrophobic, while strain GS-5 was markedly hydrophilic as compared to the other strains tested. Cell-surface protein antigens were then analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. A protein antigen of Mr 190,000 (PAc) was found in cell extracts and culture supernatants of all the hydrophobic strains. Neither culture supernatant nor cell extract of strain GS-5 contained PAc. Strain GS-5, however, produced extracellularly a large amount of a protein of Mr 155,000 (PAGS-5) which reacted with rabbit anti-PAc serum. Immunodiffusion analysis showed that PAGS-5 lacked a part of the antigenic moieties in the PAc molecule. SDS-PAGE and radioimmunoassay showed a small amount of PAGS-5 on the cell surface of strain GS-5. These findings suggest that PAGS-5 may correspond to PAc which lacks a region participating in binding of PAc to the streptococcal cell.     

Alpha 3 beta 3 complex of thermophilic ATP synthase. Catalysis without the gamma-subunit

A complex of the alpha- and beta-subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the alpha beta gamma complex. The alpha 3 beta 3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa). Dilution of the alpha beta complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the gamma-subunit. A previous method using urea for isolating the subunits resulted in an alpha beta complex with lower activity than that prepared by over-expression of the genes. The alpha beta-ATP complex was formed from the alpha beta complex, ADP and Pi in the presence of dimethyl sulfoxide.     

Mast cells are important in the development of hypersensitivity pneumonitis. A study with mast-cell-deficient mice

We have previously reported that C57B1/6 mice develop lung lesions similar to human hypersensitivity pneumonitis (HP) by repeated transnasal administration of Thermoactinomyces vulgaris antigen. Since the HP-like lesions were induced via respiratory route and by the causative antigen in human HP (farmer's lung), it seems that this murine model is useful for investigating the cell-to-cell interactions in human HP. To clarify the involvement of mast cells (MC) in the development of HP, T. vulgaris (90 micrograms/day) was transnasally administered to MC-deficient WBB6F1-W/Wv mice (W/Wv) and their littermates (+/+) five times a wk for 3 wk. When the lungs were examined by scoring pathological findings and lung indexes, HP-like lesions were significantly less severe in W/Wv than in +/+, whose lesions were equivalent to those of C57B1/6. Bone-marrow-derived cultured MC from +/+ mice (98% purity) were obtained by in vitro culture mixed with WEHI-3B-derived conditioned medium which contained IL-3. When these MC were adoptively transferred to W/Wv mice (10(7) cells/mouse), the HP-like lesions in W/Wv mice were enhanced to be as severe as those in +/+. Importantly, significant numbers of MC were found in the lungs of MC-transferred W/Wv mice. These results suggest that MC play an important role in the development of the murine experimental HP.