Intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant Sindbis virus.

A single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. To see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant Sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. The variable genes and interchain linker encoding the single-chain antibody were cloned into a double subgenomic Sindbis virus expression vector to generate recombinant Sindbis virus. Infection with this recombinant Sindbis virus provided high-level cytoplasmic expression of the antibody fragment in mammalian cells. We demonstrate (i) that the antibody fragment was antigen binding and (ii) that louping ill virus infectivity was significantly reduced in the presence of intracellular antibody expressed by the superinfecting recombinant Sindbis virus.     

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and on 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661-668). Brief exposure (1 min) to N-ethylmaleimide (0.3-10 mM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H2O2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin action was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockade on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20-30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effect of sulphydryl blockade but no protective effect was observed with H2O2. None of the oxidants protected against the inhibitory effect of 3 mM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Interaction of purified DNA with plant protoplasts of different cell cycle stage: The concept of a competent phase for plant cell transformation

Summary The polycation mediated attachment of purified tritiated DNA to plant protoplasts has been measured by quantitative microautoradiography. The automated grain counting technique used, also provides information on the cell cycle stage of individual protoplasts, which circumvents the need to synchronize the plant cell population before preparation of protoplasts. With protoplasts from asynchronously dividing suspension cultures of Nicotiana syhestris (NS-1), S-phase protoplasts appear to be inefficient binders of ³H-DNA, as compared with G₁ or G₂ protoplasts. Protoplasts derived from a tumour line of Crepis capillaris (CAPT) exhibit ³H-DNA binding at all cell cycle phases, but Sphase protoplasts appear to be preferential binders. These differences are discussed with reference to cell cycle kinetics, membrane charge variation and the possibility of increasing the efficiency of genetic transformation of higher plant cells in culture.     

A re-examination of the reaction between ox liver glutamate dehydrogenase and 1-fluoro-2,4-dinitrobenzene.

Reaction of ox liver glutamate dehydrogenase with 1-fluoro-2,4-dinitrobenzene for 4 h at pH 8 caused 86% inactivation, almost complete desensitization to allosteric inhibition by GTP, but only partial desensitization to ADP activation. The enzyme remained hexameric after such treatment. NAD⁺, but not NADH or NADPH, partially protected activity. Protection was enhanced by GTP and decreased by ADP. GTP and NADH together protected effectively, although separately neither protected. GTP and NADPH gave partial protection of activity. Glutarate and succinate, inhibitors competitive with glutamate, gave substantial protection, slightly enhanced in the presence of NAD⁺. With glutarate, but not succinate, an initial activation was seen during chemical modification. The allosteric response to GTP was protected by GTP itself only when NAD⁺ or NAD(P)H was also present; other ligands failed to protect. Similarly ADP alone did not protect ADP sensitivity. NADH partially protected ADP sensitivity, although NADPH did not. ADP itself counteracted the protection given by NADH. GTP with NADH completely protected ADP sensitivity. This combination of ligands thus protects all the assayed properties. GTP with NADPH gave less complete protection of the ADP response. Observed protection patterns varied with the pH and coenzyme concentration of the assay mixture under constant conditions of chemical modification. Overall, the results are inconsistent with the view that dinitrophenylation directly blocks nucleotide binding sites, and suggest rather that it interferes with communication between sites.     

Cell cycle analysis of tumour-derived cultures ofCrepis capillaris: A kinetic analogy with proliferation of animal tumour cells

Summary Analysis of the cell cycle by three methods has revealed unusual kinetics of proliferation in tumour derived suspensions ofCrepis capillaris. The different methods of analysis yield different estimates of cycle phase durations, and such discrepancies have been explained in terms of low growth fractions with rapid total cycle traverse. Specifically, confidence in the estimation of G₂ duration by the fraction of labelled mitosis analysis, and comparison with shorter G₂ estimates obtained by the two other methods, suggests that cells drop out in G₁. However, cells which do not drop out of the proliferative compartment traverse G₁ extremely rapidly. Extremely short cell cycle durations in which the G₁ phase is virtually non-existent are uncharacteristic of plant cell suspension cultures, in which the G₁ phase has previously been shown to be extended as compared with meristematic root tip cells. A model has been proposed in which a central core of rapidly dividing cells continuously loses cells into a subpopulation of resting or G₀ cells with the G₁ DNA content. Similarities between plant and animal tumours with respect to cell growth and division are discussed.     

Role of adrenergic innervation in experimental renal hypertension

Renal hypertension was induced by ligation of the aorta between renal arteries in rats sympathectomized with 6-hydroxydopamine. In the early phase, equally severe hypertension developed in the denervated group as compared to innervated controls. Later, blood pressure was lower in the denervated rats. Initially, increases in plasma renin were seen in both groups; the levels, however, were markedly lower in the denervated rats. Later, the renin levels were similar and not different from baseline. It is concluded that adrenergic neural activity is not essential in the development of renal hypertension; the maintenance of the chronic state, however, depends in part on adrenergic innervation.     

Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G1 transition probability model

The effects of temperature on the cell cycle of Haplopappus gracilis suspension cultures were analysed by the fraction of labelled mitoses method. Sphase in these cultures shows a different temperature optimum as compared to optima derived for G₂ and mitosis. G₁ phase has a much lower Q₁₀ than the other cell cycle phases and shows no temperature optimum between 22 and 34° C. These results are discussed in relation to a transition probability model of the cell cycle proposed by Smith and Martin (Proc. Natl. Acad. Sci. USA 70, 1263–1267, 1973), in which each cell has a time independent probability of initiating the transition into another round of DNA replication and division. The implications of such a model for cell cycle analysis are discussed and a tentative model for a probabilistic transition trigger is advanced.Abbreviations FLM Fraction of labelled mitoses - T_B Total B-phase     

Treatmenf of optic neuritis by retrobulbar injection of triamcinolone.

In a single-blind controlled clinical trial patients with optic neuritis caused by demyelination were given a single retrobulbar injection of triamcinolone. Though the treated group showed a trend towards more rapid recovery of vision than the controls, there was no significant difference in visual acuity, colour vision, or visual fields during the first six months after treatment. We conclude that routine use of corticosteroids is not justified in unilateral optic neuritis when vision in the other eye is good. Shortening the period of visual disability in bilateral disease or unilateral disease when vision in the other eye is poor, however, may be justifiable.