Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Determinants of Slow Walking Speed in Ambulatory Patients Undergoing Maintenance Hemodialysis

Walking ability is significantly lower in hemodialysis patients compared to healthy people. Decreased walking ability characterized by slow walking speed is associated with adverse clinical events, but determinants of decreased walking speed in hemodialysis patients are unknown. The purpose of this study was to identify factors associated with slow walking speed in ambulatory hemodialysis patients. Subjects were 122 outpatients (64 men, 58 women; mean age, 68 years) undergoing hemodialysis. Clinical characteristics including comorbidities, motor function (strength, flexibility, and balance), and maximum walking speed (MWS) were measured and compared across sex-specific tertiles of MWS. Univariate and multivariate logistic regression analyses were performed to examine whether clinical characteristics and motor function could discriminate between the lowest, middle, and highest tertiles of MWS. Significant and common factors that discriminated the lowest and highest tertiles of MWS from other categories were presence of cardiac disease (lowest: odds ratio [OR] = 3.33, 95% confidence interval [CI] = 1.26–8.83, P<0.05; highest: OR = 2.84, 95% CI = 1.18–6.84, P<0.05), leg strength (OR = 0.62, 95% CI = 0.40–0.95, P<0.05; OR = 0.57, 95% CI = 0.39–0.82, P<0.01), and standing balance (OR = 0.76, 95% CI = 0.63–0.92, P<0.01; OR = 0.81, 95% CI = 0.68–0.97, P<0.05). History of fracture (OR = 3.35, 95% CI = 1.08–10.38; P<0.05) was a significant factor only in the lowest tertile. Cardiac disease, history of fracture, decreased leg strength, and poor standing balance were independently associated with slow walking speed in ambulatory hemodialysis patients. These findings provide useful data for planning effective therapeutic regimens to prevent decreases in walking ability in ambulatory hemodialysis patients.     

Purification of hepatitis B virus gene X product synthesized in Escherichia coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus

A fused gene containing 94% of the hepatitis B virus (HBV) open reading frame X was expressed in Escherichia coli, and its 17-kDa product was purified by ion-exchange chromatography. Antibody elicited against the X-gene product reacted with materials proximal to the nuclear membrane of a human hepatoblastoma cell line producing HBV particles. No such reaction was observed with the same cell line that did not produce HBV particles.     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

Chromosomal mapping of 18S-28S rRNA genes and 10 cDNA clones of human chromosome 1 in the musk shrew (Suncus murinus).

The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.     

Glycyrrhizic acid and its hydrolysate as mineralocorticoid agonist

Mineralocorticoid activity of glycyrrhetinic acid (GR) was studied in vivo (electrical potential difference in rat rectum) and in vitro (brush border Mg2+-HCO3- ATPase in rat small intestine, kidney cytosol binding of GR with and without RU-28362, anti-glucocorticoid compound) in order to clarify the mechanism of mineralocorticoid-like activity of GR. Scatchard analysis of [3H]aldosterone showed that Kd of higher affinity site (type I) 6.0 X 10(-9) M, Bmax 1.0 X 10(-14) mol/mg protein, and Kd of lower affinity site (type II) 1.6 X 10(-7) M, Bmax 7.5 X 10(-14) mol/mg protein. GR competed for [3H]aldosterone binding sites in kidney cytosol at the concentration of 10(4) times as that of unlabeled aldosterone. RU-28362 displaced aldosterone binding curve, whereas GR binding kinetic was not affected by this compound. Adrenalectomy caused a significant fall in brush border Mg2+-HCO3- ATPase activity (75% reduction compared with the initial level) which was not restored by GR administration. Electrical potential differences in the adrenalecomized rats were significantly lower than those in the control rats, which did not increase after GR administration.     

Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.     

A carboxyl-terminal hydrophobic region of yeast cytochrome c1 is necessary for functional assembly into complex III of the respiratory chain

Cytochrome c1 is an amphiphilic protein which binds to the mitochondrial inner membrane, presumably through a hydrophobic region near the carboxyl (C)-terminus. In the preceding study (Hase, T., et al. (1987) J. Biochem. 102, 401-410), two cytochrome c1 mutations were constructed: delta 1 and delta 2 cytochromes c1, in which the C-terminal segments of 17 and 71 residues were replaced by foreign sequences of 20 and 15 residues, respectively. delta 2 cytochrome c1 had lost the putative membrane anchor. The two cytochrome c1 mutants were localized in mitochondria, but succinate-cytochrome c1 reductase activity was detected only in the mitochondria containing delta 1 cytochrome c1. The membrane association of the two mutant molecules as well as that of authentic cytochrome c1 was investigated. These three molecules were firmly attached to mitochondrial membranes and not solubilized on either sonication or sodium carbonate (pH 11) treatment. However, when the membranes were solubilized with Triton X-100, both the delta 1 and authentic cytochromes c1 were extracted from the membranes more easily than delta 2 cytochrome c1. By fractionating cholate extracts of mitochondrial membranes with ammonium sulfate, delta 1 cytochrome c1 was cofractionated with the enzymatic activity of complex III, but delta 2 cytochrome c1 was clearly separated from the complex III fraction. Trypsin treatment of mitochondria and mitoplasts showed that delta 2 cytochrome c1 was exposed to the intermembrane space, with such a topology that its trypsin susceptibility became much higher than that of the authentic molecule.(ABSTRACT TRUNCATED AT 250 WORDS)