Effect of free Mg2+ on liver phosphorylase kinase activity

When pig liver phosphorylase kinase was assayed at various concentrations of Mg2+, about 2-fold stimulation was observed around 2-3 mM Mg2+ (Mg2+/ATP ratio, 20-30) compared with the activity at 0.3 mM Mg2+ (Mg2+/ATP ratio, 3). This stimulation was specific for Mg2+ among the divalent cations tested and the process was reversible. Km values for ATP and phosphorylase b were decreased 3.6- and 9.5-fold, respectively, at 3 mM Mg2+ compared with those obtained at 0.3 mM Mg2+. These results indicate that the activity of liver phosphorylase kinase is influenced by free Mg2+.     

Molecular cloning of cDNA for rat acyl-CoA oxidase

Poly(A+) RNA was prepared from hepatic free polysomes of rats which had been fed di(2-ethylhexyl) phthalate for the induction of peroxisomal beta-oxidation enzymes. This preparation was enriched for the mRNAs of these enzymes by sucrose density gradient centrifugation, and used for the synthesis of double-stranded cDNA. Recombinant plasmids were constructed from the cDNA and pBR322 by dG X dC-tailing method and used for the transformation of an Escherichia coli strain, chi 1776. By differential colony hybridization using [32P]cDNA of partially purified liver poly(A+) RNA from induced and noninduced rats as probes, and then by hybridization-selected translation, we obtained two clones with cDNA inserts which specifically selected acyl-CoA oxidase mRNA. On Northern blotting, both cDNA inserts hybridized to 3.8-kilobase RNA which was increased about 10-fold by di(2-ethylhexyl) phthalate treatment of the rats. The cleavage maps of the cDNA inserts showed they overlap with each other. We conclude that the above two recombinant plasmid clones contain cDNA sequences for rat acyl-CoA oxidase.     

A new type of glycogen storage disease caused by deficiency of cardiac phosphorylase kinase

A five-month-old Japanese boy was found to have marked glycogen accumulation only in the heart. A survey of enzymes revealed normal activities of phosphorylase, cyclic AMP-dependent protein kinase, acid maltase and amylo-1,6-glucosidase. However, the heart had capacity of activating neither rabbit muscle phosphorylase b nor endogenous phosphorylase b, which was converted to active form only when supplemented rabbit muscle phosphorylase kinase. In contrast to the heart, activities of phosphorylase kinase were found within normal levels in other organ tissues so far tested. These findings indicate that the present case of the cardiac glycogenosis is caused by deficiency of cardiac phosphorylase kinase.     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.     

Modification of susceptibility to Klebsiella pneumoniae during murine cytomegalovirus infection

The effect of murine cytomegalovirus (MCMV) infection on susceptibility to bacterial infection was studied in mice by a combination of intraperitoneal (ip) inoculation of a sublethal dose of MCMV with subsequent ip challenge of 2 X 10(3) cfu of a strain of Klebsiella pneumoniae (KP). When given alone, KP produced a mortality of 30-40%. Mortality was increased when KP was given 1 to 7 days after MCMV injection with the peak increase at the 4th to 5th day when 100% mortality occurred. Virus levels in various organs of mice infected with MCMV alone, or superinfected with KP did not differ. Bacterial counts on the other hand, showed that increased mortality in mixed MCMV and KP infected mice was due to an uncontrolled growth of bacteria at the site of primary lodgment, i.e., the peritoneum, and severe systemic infection. Neutrophil response to growth of KP during the first 3 days of bacterial infection was defective in MCMV infected mice. While the initial clearance of KP from the blood was more efficient in MCMV infected mice, probably due to activated reticuloendothelial function, it did not protect the mice against KP infection. Using the in vivo model, it was shown that poor neutrophil response and possibly other defective neutrophil functions were responsible for increased mortality to KP infection in MCMV infected mice.     

Isolation of virus causing hemorrhagic fever with renal syndrome (HFRS) through a cell culture system

Twenty-three rat lung specimens collected in outbreaks of hemorrhagic fever with renal syndrome (HFRS) in three medical institutions were inoculated onto the VERO-E6 cell monolayers. After several blind passages, an agent growing serially in the cell cultures and reacting specifically with known HFRS-positive sera was isolated from two of these specimens. The two isolates were antigenically identical each other. The agent, named strain SR-11, was identified as the causative virus of HFRS by its antigenic identity with E6 cell-adapted HFRS virus, Hantaan 76-118 strain, and the specific reactions with sera from various HFRS cases.     

Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria

ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.     

Is the Fatty Acid Composition of Phospholipids Important for the Function of the Circadian Clock in Neurospora crassa?

Effects of pheriylmethanol (PM), 2-phenylethanol (PE) and 3-phenyl-1-propanol(PP) on the conidiation rhythm and on the fatty acid compositionof phospholipids were examined in Neurospora crassa. The periodfor the conidiation rhythm was shortened in proportion to theconcentration used of PE of more than 1 mM. PM at 10 mM andPP at 3 mM, however, did not cause a decrease in the lengthof the period. PE did not affect the temperature compensationof the length of the period or the amplitude of the phase shiftby light. The ratio of linolenic acid to Hnoleic acid decreased in proportionto the concentrations of PE, PM, and PP in all the phospholipidsexamined. The proportion of phosphatidic acid+diphosphatidylglycerolto the total phospholipids also was decreased by these compounds.Period shortening by PE can not be explained by the change inthe phospholipid fatty acid composition. (Received April 18, 1983; Accepted June 22, 1983)