Outermost-cell-surface changes in an encapsulated strain of Staphylococcus aureus after preservation by freeze-drying.

The effects of drying time during freeze-drying on the outermost cell surface of an encapsulated strain of Staphylococcus aureus S-7 (Smith, diffuse) were investigated, with special attention paid to capsule and slime production. To quantify capsule and slime production, capsule antigen production and cellular characteristics such as growth type in serum-soft agar, cell volume index, and clumping factor reaction were examined. After freeze-drying the colonial morphology of strain S-7 was altered from a diffuse to a compact type in serum-soft agar. In accordance with these changes, the titer of the clumping factor reaction increased while the cell volume index, capsule and slime production, and capsule antigen production were markedly decreased in parallel with the period of freeze-drying. The ability of the strain to adhere to collagen, fibrinogen, and soybean lectin was also compared before and after freeze-drying. Fibrinogen levels slightly increased when 10% skim milk and 2% honey were used as cryoprotective agents and showed a remarkable increase when 0.05 M phosphate buffer was used as a control. Also, the ability of strain S-7 to adhere to soybean lectin declined, whereas no changes were observed for collagen under any conditions. Strain S-7 was phage nontypable before freeze-drying but the number of typable cells increased after freeze-drying; phage-typable cells reacted to phage 52 alone after 5 h of freeze-drying, but additional cells also proved to be phage typable to phage 42E after 10 h. Electron micrographs indicated that strain S-7, an encapsulated strain, was converted to an unencapsulated state after freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

A new class mutation of low density lipoprotein receptor with altered carbohydrate chains

In a monensin-resistant mutant (Monr-31) of Chinese hamster ovary cells, the O-linked sugar chains of the low density lipoprotein (LDL) receptor are altered, suggesting a mutation at a Golgi apparatus gene. In a compactin-resistant mutant (MF-2) of Chinese hamster V79 cells, the mature LDL receptor is apparently 5000 daltons smaller; the difference is due to altered glycosylation of O-linked sugar chains. Hybrids between MF-2 and Monr-31 still produced LDL receptor molecules with aberrant sugar chains; thus both mutants are in the same complementation group. Krieger and his colleagues (Krieger, M., Kingsley, D., Sege, R., Hobbie, L., and Kozarsky, K. (1985) Trends. Biochem. Sci. 10, 447-452) have classified Chinese hamster ovary cell mutants with altered LDL receptor structure into four groups: ldlA, ldlB, ldlC, and ldlD. Cell-cell hybrids between their ldl mutants and Monr-31 produced wild type mature LDL receptors with normal molecular sizes, suggesting that these compactin- and monensin-resistant mutants define a new class of LDL receptor mutant. Since both of our mutants are defective in internalization of LDL, we assign them as int mutants. This may imply a further etiology for hypercholesterolemia, and cases can now be examined for such a class.     

Multiplication of Legionella pneumophila Philadelphia-1 in cultured peritoneal macrophages and its correlation to susceptibility of animals

Intracellular growth of Legionella pneumophila Philadelphia-1 strain in peritoneal macrophages (PMP) from various rodents was measured and its correlation to the level of susceptibility of the animal was examined. In guinea pig PMP, the organism grew well and the guinea pig was very susceptible to it (50% lethal dose, LD50 = 7.6 X 10(4)). On the other hand, the bacteria hardly multiplied in mouse PMP and the animal was resistant to infection (LD50 = 6.7 X 10(7)). Intracellular growth rate correlated well with susceptibility in these animals. In golden hamsters, a discrepancy between intracellular growth and susceptibility was found. The organism grew intracellularly as rapid as in guinea pig PMP, but the golden hamster was very resistant to infection (LD50 = 2.2 X 10(8)). In rat PMP, the organism did not grow intracellularly during a 24-h period of infection, but started to grow after that and the growth rate thereafter was as rapid as in guinea pig PMP. WKA rats were resistant and the LD50 in the animal was 1.9 X 10(7). In vivo natural resistance of rats and golden hamsters to the organism was considered to be a result of other factors than macrophages.     

Effect of changes in thyroid state on metabolism of thyroxine by rat placenta

We studied the effect of the state of the thyroid on T4 monodeiodination in the rat placenta, and it was compared with those in the liver and kidney. The tissues, maternal serum, and amniotic fluid were obtained from pregnant rats. The tissues were homogenized in cold 50 mM Tris-HCl buffer, pH 7.5. The homogenate (1 mg protein) was incubated at 37 degrees C for 60 min with 1 microgram T4 in the presence of 5 mM DTT. The T3 and reverse T3 generated in the reaction mixture were extracted into cold ethanol and measured by RIAs. The conversion of T4 to reverse T3 in rat placenta was not significantly changed in MMI-induced hypothyroidism or T4 induced hyperthyroidism. On the other hand, conversion of T4 to T3 in the liver and kidney were changed in parallel with the thyroid state. The concentration of reverse T3 in the amniotic fluid was increased in accordance with the increase in the maternal serum T4 concentration. These results indicate that the placental T4 inner ring deiodination is not affected by the thyroid state, and that the change in the amniotic fluid reverse T3 concentration in this study is mainly dependent upon the change in maternal thyroid function.     

Existence of glucose-6-phosphate dehydrogenase-like locus on chromosome 17.

Hybridization of DNA samples prepared from flow-sorted human chromosomes with a cDNA probe for the X-linked glucose-6-phosphate dehydrogenase (G6PD) suggested the existence of the G6PD-like locus on chromosome 17. Southern hybridization analysis of endonuclease-digested DNA samples from the human-mouse hybrid cell line with human chromosome 17, and from control human and mouse cells, proved that not only X chromosomes, but also chromosome 17, contain DNA sequences that are hybridizable with the G6PD cDNA probe. The G6PD-like locus on chromosome 17 could be a putative pseudogene or a functional gene for the fetal brain-specific G6PD isozyme or other protein.     

Reverse Changes in Plasma Membrane Properties upon Deacclimation of Mulberry Trees (Morus bombysis Koidz.)

To gain more insight into the relation between plasma membranechanges and cold hardiness in mulberry trees (Morus bombysisKoidz. cv Goroji), biochemical and biophysical changes in theplasma membrane were studied during cold deacclimation in spring.The majority of the changes in the plasma membranes that occurredduring the cold acclimation process in the fall/winter werereversed following deacclimation in the spring. Significantdecreases in phospholipid content, degree of unsaturation inphospholipid fatty acids, and membrane fluidity were observedin the plasma membranes during cold deacclimation. The sterolto phospholipid ratio increased with decreasing cold hardiness.Reverse changes were also detected in the majority of proteinand glycoprotein components. These reversible changes in theplasma membranes are considered to be involved in the mechanismof cold hardiness of plants. ¹Contribution No. 2766 from the Institute of Low TemperatureScience. (Received July 10, 1985; Accepted October 25, 1985)     

Effects of the calcium channel blocker diltiazem on the excitability of mouse oocytes

Effects of the organic Ca antagonist diltiazem on Ca channels were studied in ovulated and unfertilized oocytes of the mouse by using intracellular recording techniques. The resting potential was not affected by diltiazem. The threshold level of the Ca action potential shifted slightly toward positive voltages with diltiazem concentration, but the shift was not statistically significant. The overshoot and maximum rate of rise of the Ca action potential were inhibited by the drug in a dose-dependent manner, but higher amounts of diltiazem were necessary to cause similar blocking effects on Ca channels in mouse oocytes than in other differentiated cells. Increases of external concentration of Ca²⁺ antagonized the degree of diltiazem inhibition. However, the sequence of block of Ca²⁺, Sr²⁺, and Ba²⁺ currents was different for diltiazem vs Cd²⁺. It is suggested that diltiazem inhibition can not be explained by simple competitive scheme, ie, antagonism between diltiazem and permeant cations does not occur at the same binding site associated with the Ca channel in mouse oocytes.