Effect of temperature on the velocity of erythrocyte aggregation

The velocity of the aggregation of human erythrocytes was examined in the range of 5-43 degrees C with a rheoscope combined with a video camera, an image analyzer and a computer. (1) With increasing temperature, the velocity of erythrocyte aggregation induced by fibrinogen, immunoglobulin G and artificial macromolecules (dextran of 70 kDa and poly(glutamic acid) of 50 kDa) increased. However, the relationship between the velocity of erythrocyte aggregation and the temperature was different among these macromolecules. (2) In 70% autologous plasma, the velocity of erythrocyte aggregation was minimum at 15-18 degrees C, and increased at both higher and lower temperatures. (3) The shape of erythrocyte aggregates in 12 mumol/l fibrinogen (containing 770 mumol/l albumin) and in 70% autologous plasma was dependent on temperature: three-dimensional below 15-18 degrees C and one-dimensional (mainly rouleaux) above 15-18 degrees C. However, the shape of aggregates in 27 mumol/l immunoglobulin G (containing 770 mumol/l albumin) was three-dimensional in all temperature ranges. (4) The temperature dependency of erythrocyte aggregation was discussed in terms of the changes of medium viscosity, of erythrocyte properties and of bridging macromolecules.     

Assignment of the apolipoprotein A-I gene to 11q23 based on RFLP in a case with a partial deletion of chromosome 11, del(11)(q23.3→qter)

Summary The XmnI genotype at the apolipoprotein A-I locus was heterozygous in a boy with partial deletion of the long arm of chromosome 11, del(11)(q23.3qter). The apolipoprotein A-I gene, previously assigned to chromosome region 11q23q24, has been more specifically localized to 11q23 by excluding the region 11q24qter.     

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.     

Effects of compound eye-removal on the photoperiodic response of the band-legged ground cricket, Pteronemobius nigrofasciatus

The band-legged ground cricket Pteronemobius nigrofasciatus shows a clear photoperiodic response at 25°C with respect to the control of the induction of embryonic diapause. When crickets were reared under a short-day (LD 12 12) photoperiod and then transferred to a long-day (LD 16 8) photoperiod upon adult emergence, the adults mainly laid nondiapause eggs. However, adults maintained continuously under short-day conditions laid dispause eggs. When compound eyes were bilaterally removed after adult emergence, the crickets mainly laid nondiapause eggs, irrespective of the photoperiod. Thus, the adults completely lost their sensitivity to photoperiod after bilateral removal of their compound eyes. Unilateral removal of the compound eye also affected the crickets under a short-day photoperiod, and the incidence of diapause eggs was intermediate between that laid by intact adults and that laid by adults after the bilateral removal of compound eyes. The incidence of diapause eggs in sham-operated crickets was not significantly different from that in intact crickets under both sets of photoperiodic conditions. These results show that P. nigrofasciatus perceives the photoperiod through its compound eyes.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Resonance Raman spectra of riboflavin and its derivatives in the bound state with egg riboflavin binding proteins

The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7,8-dichlororiboflavins (7,8-Cl RF), in H2O and D2O were observed in the 700-1700 cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm-1 line of RF was shifted to ca. 1300 cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm-1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C = O stretching mode was observed in the present resonance Raman spectra.     

A resonance Raman study on a reaction intermediate of Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i.e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of D-amino acid oxidase (DAO). The isotope effects on the 1,665 cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C = N stretching mode of an imino acid derived from phenylalanine, i.e., alpha-imino-beta-phenylpropionate. The intense band at 1,389 cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of alpha-imino-beta-phenylpropionate. The 1,602 cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1,605 cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10a) = C(4a) - C(4) = O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a) = C(4a) - C(4) = O of reduced flavin and the plane containing H2+N = C - CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.     

The primary structure of rat rig/ribosomal protein S15 gene.

We have isolated rat rig/ribosomal protein S15 gene from a DNA library derived from a rat insulinoma and determined the complete nucleotide sequence. The rat rig/S15 gene is composed of four exons and three introns spanning 2 kbp and exhibits distinctive structural features unique for a ribosomal protein gene.     

Proton release from flavoprotein D-amino acid oxidase on complexation with the zwitterionic ligand, trigonelline

Trigonelline, i.e., N-methylnicotinate, which has a zwitterionic structure similar to a substrate D-amino acid, is a useful active site probe for D-amino acid oxidase (DAO). The affinity of trigonelline for DAO in the deprotonated state at the enzyme bound FAD 3-imino group is higher than in the neutral state, contrary to in the case of benzoate, which is a competitive inhibitor and is in a monoanionic form. The time course of the absorbance change was monitored for the binding of DAO with trigonelline by means of a stopped-flow technique. The reaction, on monitoring at 507 nm, was found to be biphasic at pH 8.3, with fast and slow phases. The dissociation of the 3-imino proton of the enzyme bound FAD was observed in the same time course as the slow phase. These results suggest that the positive charge of trigonelline exists near the 3-imino group of the enzyme bound FAD and interacts repulsively with the proton of the 3-imino group. The absorption spectra of the DAO-trigonelline complex at various pHs also support this hypothesis. In the catalysis of DAO, a similar mechanism may be involved, that is, the positive charge of a D-amino acid may interact repulsively with the 3-imino proton of the enzyme bound FAD, and this interaction may be important for the catalysis.     

Arginase as an inhibitory principle in liver plasma membranes arresting the growth of various mammalian cells in vitro

Plasma membranes prepared from rat livers inhibited the in vitro growth of various mammalian cells including hepatoma cells in a concentration-dependent manner, showing an almost complete arrest of cell growth at 0.1 mg protein/ml. Some of these cells tested, i.e., leukemia (L1210 and P388) and myeloma (P₃-NS-1/1-Ag4-1) cells, were labile in the presence of plasma membranes (losing the viability), and CHO (Chinese hamster ovary) cells became round without detaching from the substratum. The culture medium preincubated with liver plasma membranes no longer supported the growth of hepatoma cells (AH13 and AH66F). However, the ‘conditioned’ medium supplemented with l-arginine, supported the growth of the cells. Moreover, the addition of l-ornithine to the cultures containing plasma membranes markedly reduced the inhibitory effect of plasma membranes. The plasma membrane preparations were found to possess considerable arginase activity. These results seem to indicate the possible involvement of arginase in the inhibition of cell growth by liver plasma membranes.