Oligoribonucleotide initiators for herpes simplex virus DNA synthesis in vivo and in vitro.

Replicating herpes simplex virus (HSV) DNA, labeled with [³H]thymidine or [³H]uridine, was isolated from infected primary rabbit kidney cells. Nascent DNA strands are covalently linked to oligoribonucleotide stretches. The chain length of this RNA segment amounts to approximately 36 nucleotides wich are located at the terminus of the replicating DNA. The extent of RNA initiator synthesis seems to be closely correlated with the rate of HSV-DNA synthesis. The half life of the RNA initiator was estimated as 35 min. Evidence is presented by comparative studies regarding the characteristics of the naturally occurring RNA initiator, that cordycepin (3′-dAdo) is an inhibitor of the synthesis of the RNA initiator: (1) identical maximal chain length of the 3′-dAMP-RNA, (2) identical half-life of the 3′-dAMP-RNA segment, and (3) susceptibility to RNases and alkali. The 3′-dAMP-RNA segment is not covalently linked to HSV-DNA. HSV-Induced DNA polymerases utilizes not only deoxyribooligomers $\text{[}\text{d(pA)}{}_{\mathrm{<mtext>8</mtext>}}\text{]}$ as initiators for the DNA template-dependent DNA polymerization. The initiating activity of both the DNA and the RNA segment is abolished when it carries a 3′-dAMP moiety at its 3′-terminus.     

Hybridization studies of the relationship between influenza virus RNA and cellular DNA.

The possibility that an influenza RNA component was transcribed from cell DNA was examined by molecular hybridization with fowl plague virus (FPV) RNA and denatured chick cell DNA. RNA from purified preparations of FPV formed stable hybrids with chick DNA immobilized on nitrocellulose membranes. This RNA was located within virus cores produced by bromelain treatment and had properties characteristic of ribosomal RNA. To detect unique gene products in FPV-RNA and to determine the extent of homology, hybridization was carried out in solution under conditions where the denatured chick DNA was in vast excess. The kinetics of hybridization of purified FPV-RNA and chick rRNA with this DNA were compared. Two to eight percent of RNA from purified virus hybridized to DNA with a $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ characteristic of rRNA. Chick rRNA also competed effectively with viral RNA-DNA hybrid formation. It was concluded that influenza viruses encapsidate variable amounts of rRNA; no evidence was obtained for the existence of unique cell gene products in influenza virus RNA.     

Purification and some properties of beet yellow virus

Beet yellows virus was purified by a method, based on ultracentrifuging at 35,000 g, that preserved the normal length of the virus particle and eliminated a uv-absorbing contaminant retained by previously described methods. The purified particles had a modal length in shadowed preparations of 1250 nm and sedimented in the analytical centrifuge usually as one component at 130 S. Purified preparations had an $\text{A}{}_{260}\text{A}{}_{280}$ ratio of 1.44 and an extinction coefficient at 260 nm of 2.9. In Cs₂SO₄ isopycnic banding, the virus had a density of 1.285 g/cm³, while in CsCl two bands were produced at 1.307 and 1.312 g/cm³.     

Gustatory reaction time under variable stimulus parameters in human adults

Reaction times and perceived intensities for NaCl solutions were measured in sixteen human adults. Stimulus delivery was by means of a circular piece of solution-soaked filter paper held with forceps. The reaction time (T) decreased and the perceived intensity (S) increased with increasing the concentration (C) of NaCl solution applied to a fixed (78.5 mm²) tongue area. The comprehensive relations among T, S and C can be expressed by the following formulae; $\text{T = a +}\text{b}\text{log(}\text{C}\text{C}{}_0\text{)}$, $\text{T = p +}\text{q}\text{log(}\text{S}\text{S}{}_0\text{)}$, and logS = mlogC + logn, where a, b, p, q, m, n = constants, and S₀ indicates the perceived intensity at the threshold concentration (C₀). Meanwhile, the reaction time decreased and the perceived intensity increased with increasing the stimulated area (A) under a fixed (1.0 M) concentration of NaCl solution. The relations among T, S and A can also be expressed by the following equations; $\text{T = a′ +}\text{b′}\text{log(}\text{A}\text{A}{}_0\text{)}$, $\text{T = p′ +}\text{q′}\text{log(}\text{S}\text{S}{}_0\text{)}$, and logS = m′logA + logn′, where a′, b′, p′, q′, m′, n′ = constants, and A₀ indicates the threshold size of stimulated area.     

Circadian rhythms in the rat: Constant darkness,entrainment to T cycles and to skeleton photoperiods

Free running activity and drinking rhythms of male Sprague-Dawley rats were observed in constant darkness (DD) for up to 44 days. The average period of the rhythms ($\text{τ}{}_{\mathrm{<mtext>dd</mtext>}}$) was 24.2 hr (±0.12 hr) and the activity time was near one half of the circadian cycle. In the second experiment, rats were entrained to T cycles (T=period) with 2 hr of light per cycle. At T=23 and T=26 about one half of the rats entrained indicating that these periods are near the limits of entrainment. T=23 induced a lasting aftereffect on $\text{τ}{}_{\mathrm{<mtext>dd</mtext>}}$ while T=26 affected $\text{τ}{}_{\mathrm{<mtext>dd</mtext>}}$ only briefly. In contrast to some other nocturnal rodents, activity time was not compressed as T neared the limits of entrainment. In the third experiment, rats and hamsters were entrained to 24 hr skeleton photoperiods (two 1 hr light pulses/cycle). Rats phase jumped to the longer subjective night when the interval between the light pulses was reduced to 6 or 5 hr, while most hamsters phase jumped at 3.5 hr. Furthermore, all rats phase jumped by means of delaying transients while most hamsters showed advancing transients. Finally, while skeleton photoperiods compressed activity time in hamsters to 6 hr or less, activity time remained fairly constant in rats. These results demonstrate considerable differences in the organization of the circadian system among commonly studied nocturnal rodents.     

Independent polypeptide chain initiation sites for the synthesis of different classes of proteins for an RNA tumor virus: mouse mammary tumor virus.

To distinguish the synthesis of mouse mammary tumor virus (MMTV) structural proteins on a common precursor polypeptide from their independent synthesis on separate ribosome initiation sites, we have employed the NaCl hypertonic shock technique which differentially inhibits polypeptide chain initiation on different classes of messenger RNA in eukaryotic cells. Using this method to inhibit polypeptide initiation in the MMTV-producing C3H mouse cell line $\text{Mm5mt}\text{c}{}_1$, we have selectively inhibited the synthesis of MMTV glycosylated proteins (gp52 and gp36) relative to MMTV nonglycosylated proteins (p28, p22, p18, p14, p10). These results support the concept of independent polypeptide chain initiation sites for the synthesis of these two classes of MMTV proteins. Furthermore, the use of this technique provides evidence for the lack of strict coordinate synthesis of these proteins in an MMTV-infected cell.     

High rates of excitatory miniature currents in crayfish claw opener muscle evoked by high concentrations of gamma-aminobutyric acid (GABA) in normal and Ca2+-deficient superfusions

High concentrations (0.5 mol/l) of the neutral amino acid GABA were used to evoke release of transmitter quanta from excitatory terminals at voltage clamped crayfish muscle fibres in normal and Ca²⁺-deficient superfusions. An experiment in which the release of transmitter quanta proceeded at high rates in both normal and Ca²⁺-deficient superfusion was analyzed in detail indicating a Ca²⁺-independent mechanism of release. In the normal superfusion, on application of GABA, the release rates ñ increased within a few seconds up to about 6000 quanta/s and thereafter declined exponentially with a time constant $\text{τ}{}_{\mathrm{<mtext>q</mtext>}}\text{) = 18.5}\text{s}$, most likely due to depletion of a readily releasable store of transmitter in the excitatory nerve terminals comprising at least 110,000 quanta per muscle fibre. Assuming that about 1900 excitatory synapses exist per muscle fibre [9], it results that about 58 quanta can be associated with each synapse in agreement with morphological data [15] which show that between 47–117 vesicles exist in a single glutamatergic synapse of crayfish.     

Effects of glucosamine, 2-deoxyglucose,and tunicamycin on glycosylation,sulfation, and assembly of influenza viral proteins

The effects of glucosamine, 2-deoxy-D-glucose (2-DG), and tunicamycin (TM) on influenza viral glycoproteins was investigated. The electrophoretic mobility of the hemagglutinin (HA) polypeptide progressively increased with increasing concentration of glucosamine, accompanied by a decrease in the incorporation of [³H]fucose into the glycoprotein. However, a significant amount of the label was detected in association with HA even when cells were treated with glucosamine concentrations as high as 40 mM In this case, the peak of [³H]fucose label associated with HA was distinct from HA₀ (HA in which glycosylation is maximally inhibited by a given inhibitor)² detected with labeled amino acid precursors; the former showed slower electrophoretic mobility than the latter, indicating that HA glycoproteins produced in the presence of glucosamine are heterogeneous with respect to the extent of glycosylation. Analysis of the smooth cytoplasmic membrane fraction showed that a significant amount of ${}^{35}\text{SO}{}_4{}^{\mathrm{?2}}$ was also incorporated into HA polypeptides synthesized in cells treated with 40 mM glucosamine; the peak of ³⁵S label coincided with that of [³H]fucose label rather than HA₀. When cells were treated with increasing concentrations of 2-DG, the incorporation of ${}^{35}\text{SO}{}_4{}^{\mathrm{?2}}$ 2 into HA decreased in parallel with that of [³H]glucosamine, and no incorporation of either label was detected in cells treated with 10 mM 2-DG. TM completely inhibited the incorporation of [³H]glucosamine into viral glycoproteins at a concentration as low as 0.29 μM. Although HA₀ was not resolved in whole cells treated with TM, it was clearly resolved in isolated smooth membrane fractions. No ${}^{35}\text{S0}{}_4{}^{\mathrm{?2}}$ label was detected in the HA₀ protein. The presence of HA₀ in smooth membranes of TM-treated cells suggests that neither glycosylation nor sulfation is essential for membrane association or migration of HA from rough to smooth membranes. It also appears that such modifications of HA are not required for integration of the protein into plasma membranes since HA₀ was detected in plasma membranes isolated from both 2-DG and TM-treated cells. Formation of virus particles was observed in the presence of 2-DG, although the yield was markedly reduced. Such particles were found to possess aberrant glycoproteins, and their hemagglutinating activity was significantly lower than that of control virions. Particles without hemagglutinating activity were also produced by TM-treated cells. Analysis of polypeptides of these particles showed that they contain unglycosylated proteins but lack any detectable glycoproteins.     

A pocket calculator program for the evaluation of the oxygen diffusion to perfusion ratio

A pocket calculator program has been written for the evaluation of the oxygen difusion to perfusion ratio (). It allows one to avoid the computational errors involved by approximating the oxygen dissociation curve through a straight line.     

A calcium regulated adenosine triphosphatase in Entamoeba histolytica

Axenically grown trophozoites of Entamoeba histolytica (NIH-200 strain) contain an active ATPase (170 nmol PO₄/min per mg protein) with maximal activity at pH 8.8, a high affinity for ATP ($\text{K}{}_{\mathrm{m}}\text{? 40 μM}$) and an absolute and specific requirement for Ca²⁺. The activation by Ca²⁺ shows positive cooperativity (n_H = 2.48) at calcium concentrations below 8 μM and no cooperativity between 8 and 25 μM. The latter concentration fully saturates the enzyme. The observed activity is insensitive to oligomycin, ouabain and ruthenium red and is unaffected by a range of inhibitors of electron transport and uncouplers of oxidative phosphorylation. The enzyme exhibits structure bound latency and is tightly bound to cellular membranes. It is sedimentable (>80%) by high speed centrifugation of cell homogenates which are either protected osmotically or in which subcellular structures are damaged by sonication or treatment with Triton X-100. Arrhenius plots of V in the temperature range of 0–38°C are linear without breaks, similar to other pyrophosphatases of E. histolytica. The calculated activation energy is 14.8 kcal/mol. This finding as well as the failure of phospholipase treatment to affect activity indicate that interactions with membrane lipids play no role in the catalytic function of this tightly membrane-bound ATPase.     

The ribonucleic acids of Uukuniemi virus,a noncubical tick-borne arbovirus

Sucrose gradient centrifugation of SDS-treated, purified Uukuniemi virus labeled with ${}^{32}\text{P}$, gave three reproducible peaks of single-stranded RNA, which sedimented at 27–29 S (L), 22 S (M), and 17–19 S (S). In polyacrylamide-agarose gels these RNAs separated into four peaks with apparent molecular weights of about 4.1 × 10⁶ (L), 1.9 × 10⁶ (M), 0.88 × 10⁶ (S1), and 0.78 × 10⁶ (S2), respectively. The major M and L RNAs, which represented about 75% of the total, were found in a constant molar ratio of 1:4.6.Treatment of the RNA species with 1 M formaldehyde or by heating to 100 ° did not result in a degradation of the larger to the smaller RNAs.The base compositions of the RNA species did not differ significantly from each other, but were clearly different from those of the ribosomal 28 S and 18 S RNAs extracted from host chick embryo cells.RNAs with identical sedimentation values and mobilities in gels were found from Uukuniemi virus-infected cells labeled with [${}^3\text{H}$]uridine in the presence of actinomycin D.     

Response characteristics of a multicompartment frog skin epidermis model.

The main goal of this study was to analyze by computer simulation the responses of a multicompartment frog skin epidermis model to systematic changes in the rate coefficients (k's) which characterize the pathways of flow of Na⁺ between compartments. Responses were expressed as deviations from the behavior of a reference model (10Es) which closely simulates known laboratory data on skin epidermis. This includes transepidermal flux rates, Na⁺ pool sizes, isotope labeling fraction, efficiency of the transepidermal Na⁺ pump ($\text{ΔNa}{}^{\mathrm{+}}\text{ΔO}{}_2$). The response patterns of 10Es with its k-variants may be useful in laboratory studies aimed at localizing the effects of an applied physical or chemical stimulus on maintenance Na⁺ metabolism, or on Na⁺ fluxes in frog skin.     

Protein synthesis in virus-infected plants. 3. Effects of tobacco mosaic virus mutants on protein synthesis in Nicotiana tabacum

Nicotiana tabacum, infected with three widely differing chemically produced and one natural mutant of tobacco mosaic virus (TMV), was pulse labeled with [³H]leucine. Uninfected plants were similarly pulse labeled with [¹⁴C]leucine. The ³H- and ¹⁴C-labeled leaf tissues were combined, fractionated into subcellular fractions, denatured, dialyzed, and electrophoresed on polyacrylamide gels in SDS. Increases or decreases in the ${}^3\text{H}{}^{14}\text{C}$ ratio in gel slices indicated changes in the proteins synthesized in the infected fractions as compared to the uninfected control fractions.In contrast to wild-type TMV, where only coat protein was detected (Singer, 1971), protein synthesis differed markedly among the mutants and their subcellular fractions in both the number and molecular weights of the protein peaks. No protein was found to be common to all strains in the same subcellular fractions except the coat protein. In addition, decreases in the ${}^3\text{H}{}^{14}\text{C}$ ratio were found, indicative of suppression of specific host proteins. The total molecular weights of proteins appearing upon infection with some mutants exceeded the coding capacity of the viral nucleic acid, and there was no indication that any of the very large proteins found were precursors. It appears probable that these multiple responses specifically produced by mutants are the result of changes in host protein synthesis caused by the infection.     

Formation of the guanylylated and methylated 5′-terminus of vaccinia virus mRNA

mRNA was synthesized in vitro by vaccinia virus particles in the presence of S-adenosyl-[methyl-³H]methionine and α- or β,γ-³²P-labeled ATP or GTP. The two 5′-terminal structures m⁷G(5′)ppp(5′)G^m and m⁷G(5′)ppp(5′)A^m were isolated after P₁ nuclease digestion of the RNA. The distribution of radioactivity between the methylated mononucleotides and inorganic phosphate released by nucleotide pyrophosphatase treatment of m⁷G(5′)ppp(5′)G^m and m⁷G(5′)ppp(5′)A^m was consistent with the formation of the latter structures by condensation of pG residues from GTP with ppG- and ppA- at the 5′-termini of vaccinia mRNAs. An alternative mechanism involving the transfer of ppG residues to pA- at the 5′-terminus of the RNA was ruled out by the ³²P-labeling data as well as by the formation of m⁷G(5′)ppp(5′)A^m- when β,γ-methylene-GTP was substituted for GTP. At limiting concentrations of S-adenosylmethionine, the predominant methylated 5′-terminal structure was m⁷G(5′)ppp(5′)N- in which the penultimate nucleoside was unmethylated; in the absence of S-adenosylmethionine, G(5′)ppp(5′)N- as well as unblocked ppN- termini were detected. On the basis of the structures formed under various conditions, the following sequence of reactions was considered:

${}^{\mathrm{\gamma \beta \alpha }}\text{ppG}\text{+}{}^{\mathrm{\beta \prime \alpha \prime }}\text{ppN}\text{?→}\text{G}\text{(5′)}\text{N}\text{?+}{}^{\mathrm{\gamma \beta }}\text{PPi}$

$\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoMet}\text{→}\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoHcy}$

$\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoMet}\text{→}\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}{}^{\mathrm{m}}\text{+}\text{AdoHcy}$