Three-year survey of the epidemiology of rotavirus, enteric adenovirus, and some small spherical viruses including "Osaka-agent" associated with infantile diarrhea

Studies were made by electron microscopy (EM) on the viruses associated with diarrhea of outpatients at a pediatric clinic in Osaka Prefecture during the three year period from 1980 through 1982. The viruses detected by EM by negative staining with phosphotungstic acid (PTA) were classified morphologically into 6 groups: rotavirus, adenovirus and four kinds of small spherical viruses, calicivirus, astrovirus, picornavirus/parvovirus (P/P)-like agent and Osaka-agent. Osaka-agent seems to be a newly identified small virus. It is 35-40 nm in diameter with a fringe of spike-like structures on its surface. Viruses were detected in 181 of the 395 cases of diarrhea (45.8%). Rotavirus was detected in 122 (30.9%) of the total cases and in 67.4% of the virus-positive cases, while other viruses were detected in 15% of the total cases; adenovirus in 23 (6%) and small agents in 36 (9%). Rotavirus infection showed a distinctive seasonal variation, being mainly restricted to cooler months, but infections with other viruses did not show any seasonal variation. The age distribution of patients suggested that infants of 0 to 2 years old are very susceptible to all viruses. Attempts to cultivate these viruses in vitro were successful with only two isolates of adenovirus type 5.     

Resonance Raman study of cytochrome b562-o complex, a terminal oxidase of Escherichia coli in its ferric, ferrous, and CO-ligated states

Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli, has been studied by resonance Raman spectroscopy in its air-oxidized, dithionite-reduced, and reduced and CO-ligated states. In the reduced state, with a 406.7-nm excitation, there appeared 1494 and 1473 cm-1 lines, indicating that low spin and high spin components are included in the cytochrome b562-o complex. For the air-oxidized protein, resonance Raman lines were observed at 1372, 1503, and 1580 cm-1 with a 413.1-nm excitation, indicating that there is a ferric low spin heme. In addition, a weak but appreciable Raman line was observed at 1480 cm-1 assignable to a ferric high spin heme. Accordingly, it was concluded that low spin and high spin components are included in the cytochrome b562-o complex in the reduced and the air-oxidized states. In the CO-ligated state, with a defocused laser beam of 413.1 nm, two Raman bands assignable to the Fe-CO stretching mode have been observed at 489 and 523 cm-1, as a major and a minor component, respectively. When the laser beam was focused upon the sample to cause a photodissociation of CO from the heme moiety, the intensity of the major band at 489 cm-1 was reduced as expected. On the other hand, the minor band at 523 cm-1 remained still obvious. It was suggested that the cytochrome b562-o complex may have an additional anomalous site for CO that is resistant to photodissociation.     

15N-labeled Escherichia coli tRNAfMet, tRNAGlu, tRNATyr, and tRNAPhe. Double resonance and two-dimensional NMR of N1-labeled pseudouridine

The N1 imino units in Escherichia coli tRNAfMet, tRNAGlu, tRNAPhe, and tRNATyr were studied by 1H-15N NMR using three different techniques to suppress signals of protons not attached to 15N. Two of the procedures, Fourier internuclear difference spectroscopy and two-dimensional forbidden echo spectroscopy permitted 1H and 15N chemical shifts to be measured simultaneously at 1H sensitivity. The tRNAs were labeled by fermentation of the uracil auxotroph S phi 187 on a minimal medium containing [1-15N]uracil. 1H and 15N resonances were detected for all of the N1 psi imino units except psi 13 at the end of the dihydrouridine stem in tRNAGlu. Chemical shifts for imino units in the tRNAs were compared with "intrinsic" values in model systems. The comparisons show that the A X psi pairs at the base of the anticodon stem in E. coli tRNAPhe and tRNATyr have psi in an anti conformation. The N1 protons of psi in other locations, including psi 32 in the anticodon loop of tRNAPhe, form internal hydrogen bonds to bridging water molecules or 2'-hydroxyl groups in nearby ribose units. These interactions permit psi to stabilize the tertiary structure of a tRNA beyond what is provided by the U it replaces.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). III. Effect on cell proliferation and immune responses

Stimulated T cell-derived inhibitory factor for cellular DNA synthesis (STIF), a lymphokine produced from concanavalin A (Con A)-stimulated rat suppressor T cells, was examined for its inhibitory effect on various cultured cells and on in vitro immune reactions. STIF could inhibit the DNA synthesis of a variety of normal and neoplastic cells from rats, mice, and humans in a dose-dependent fashion. Kinetics studies revealed that STIF selectively inhibited cellular DNA synthesis after incubation for 12 hr, but after 36 hr, it also inhibited RNA and protein syntheses. The inhibited cellular DNA synthesis by 12-hr incubation with STIF was recovered after culturing the cells in STIF-free medium. The inhibitory effect of STIF on the DNA synthesis was not blocked by addition of a sugar (alpha-methyl-D-mannoside, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, L-fucose, or L-rhamnose) in culture, as determined by using rat bone marrow cells. STIF inhibited proliferative responses of rat lymphocytes to T cell mitogens, Con A and phytohemagglutinin, and a B cell mitogen, lipopolysaccharide, as well as IL 2-dependent growth of cloned T572 cells. It could also inhibit both blastogenesis and cytotoxic T cell generation in allogeneic mixed lymphocyte reaction. The release of IL 2 from Con A-stimulated T cells was also inhibited by the added STIF in culture, as demonstrated from the finding that IL 2 activity was not detected in the supernatants even after an anion-exchange column chromatography. These results indicate that STIF could inhibit cellular DNA synthesis in a species-unrestricted manner and thus inhibits the proliferation of various normal and neoplastic cells, and that it could also inhibit lectin- or IL 2-dependent T cell proliferation as well as IL 2 production from T cells in in vitro immune reactions.     

Inactivation of rice bran thiamine-binding protein by N,N'-dicyclohexylcarbodiimide

The addition of a carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) to thiamine-binding protein isolated from rice bran resulted in a remarkable loss of its binding activity with [14C]thiamine. Thiamine and chloroethylthiamine substantially protected the protein against inactivation by DCCD, whereas thiamine phosphates did not. Another carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inactivated rice bran thiamine-binding protein. Inactivation of the thiamine-binding protein was accompanied by covalent binding of DCCD to the protein as shown by the use of [14C]DCCD. The binding of [14C]DCCD to the thiamine-binding protein was specific, and significantly inhibited by the addition of thiamine. The loss of thiamine-binding activity was proportional to the specific binding of [14C]DCCD. For complete inactivation of the thiamine-binding activity, the binding of 2.46 mol of [14C]DCCD per mol of thiamine-binding protein was required. Furthermore, limited proteolysis of the binding protein by trypsin yielded two polypeptides with molecular weights of 35,000 (large polypeptide) and 12,500 (small polypeptide) which were separated by SDS-polyacrylamide gel electrophoresis. The binding sites of [14C]DCCD were found to be located on the large polypeptide. These results suggest that a specific carboxyl residue in the large polypeptide releasable from rice bran thiamine-binding protein by trypsin digestion when modified by DCCD is involved in the binding of thiamine.     

The structure of nucleosome core particles as revealed by difference Raman spectroscopy.

Raman spectra have been observed of nucleosome core particles (I) prepared from chicken erythrocyte chromatin, its isolated 146 bp DNA (II), and its isolated histone octamer (H2A+H2B+H3+H4)2 (III). By examining the difference Raman spectra, (I)-(II), (I)-(III), and (I)-(II)-(III), several pieces of information have been obtained on the conformation of the DNA moiety, the conformation of the histone moiety, and the DNA-histone interaction in the nucleosome core particles. In the nucleosome core particles, about 15 bp (A.T rich) portions of the whole 146 bp DNA are considered to take an A-form conformation. These are considered to correspond to its bent portions which appear at intervals of 10 bp.     

Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP.

The dideoxy chain termination method using deoxy-7-deazaguanosine triphosphate (dc7GTP) in place of dGTP was found to be very useful. Sequencing of a part of the human N-myc gene having 85% GC content is impossible by the original method using dGTP, because of compression of bands. However, the nucleotide sequence of this part was unambiguously determined by analysis of both strands by the modified method. Use of dc7GTP is concluded to improve the dideoxy chain termination method for DNA sequencing.     

Modification of batroxobin with activated polyethylene glycol: reduction of binding ability towards anti-batroxobin antibody and retention of defibrinogenation activity in circulation of preimmunized dogs

Amino groups of batroxobin (Bothrops atrox thrombic protease) were modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2). The modified batroxobin had the reduced binding ability towards anti-batroxobin antibody but retained its enzymic activity in vitro and in vivo. Administration of modified batroxobin in which 29% of the total amino groups in the molecule had been modified, to beagle dogs preimmunized with native batroxobin gave rise to a marked reduction of the fibrinogen level in plasma, accompanied with an increased level of fibrinogen (fibrin) degradation products, FDP. On the other hand, no reduction of fibrinogen level was observed when native batroxobin instead of modified batroxobin was injected to immunized dogs.     

Escherichia coli uvrD mutants with thermosensitive DNA-dependent adenosine triphosphatase I (helicase II)

Three mutants producing thermosensitive DNA-dependent Adenosine triphosphatase (ATPase) I were screened from a collection of temperature-sensitive mutants of Escherichia coli K12. ATPase I purified to near homogeneity from one of the mutants (JE11000) possesses both thermosensitive DNA-dependent ATPase and DNA helicase activities. We have shown that ATPase I is encoded by the uvrD gene as first suggested by Oeda et al. (1982): (i) the thermosensitive ATPase I mutation present in JE11040 lies in or very close to the uvrD gene, (ii) ATPase I activity is absent in uvrD210, uvrD156, and uvrD252 mutants. Thus the thermosensitive mutations correspond to new uvrD mutations. However, the mutation present in JE11040 confers neither UV sensitivity nor mutator phenotype at high temperature. Evidence is presented that the mutant ATPase I is stabilized in vivo at 42 degrees C.     

Effects of Net and Local Charges on the Interaction between Chemically-Modified Horse Heart Cytochrome c and P700 in Photosystem 1-Enriched Subchloroplast Particles

Effects of salt and pH on the re-reduction of P700 by chemically-modifiedhorse heart cytochrome c after a flash illumination were examinedin Triton-treated P700- enriched subchloroplast particles (TSF-1particles). At low salt concentrations net charges on the membrane surfaceand native, guanidinated or succinylated cytochrome c were majorfactors that determined the reaction rates, as in the reactionbetween plastocyanin and P700 [Tamura et al. (1981) Plant &Cell Physiol. 22: 603]. The reaction rates also depended onreactant-specific factors, particularly the localized distributionof charges on macromolecules and their interaction over shortdistances, as well as on long-range Coulombic interaction. Theeffect of this type became clearer at high salt concentrations. (Received October 7, 1982; Accepted December 20, 1982)