The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.     

Polarized endocytosis and transcytosis in the hypothalamic tanycytes of the rat

Four types of tanycytes can be distinguished in the rat hypothalamus: ₁ and ₂ tanycytes establish an anatomical link between the ventricular cerebrospinal fluid (CSF) and the arcuate nucleus, whereas ₁ and ₂ tanycytes establish a link between CSF and portal blood. Endocytosis and transcytosis in these cells have been investigated by (1) immunocytochemistry with antibodies against molecular markers of the endocytotic and transcytotic pathways; (2) the administration of wheat germ agglutinin (WGA) into the ventricular or subarachnoidal CSF and following its internalisation by and its routing through tanycytes. The four populations of tanycytes show marked differences concerning the expression and subcellular location of proteins involved in endocytosis and transcytosis, such as clathrin, caveolin-1, Rab4 and ARF6. Thus, _1,2 tanycytes express caveolin-1 at the ventricular cell pole and at their terminals contacting the portal capillaries, whereas _1,2 tanycytes do not, suggesting that caveolae-dependant endocytosis does not occur in the latter and that, in _1,2 tanycytes, it may occur at both cell poles. In _1,2 tanycytes, clathrin is only expressed at the ventricular cell pole indicating that clathrin-dependant endocytosis operates for compounds present in the ventricular CSF and not for those exposed to the terminals. This agrees with the property of _1,2 tanycytes of internalising WGA through the ventricular cell pole but not through the terminals. The subcellular distribution in _1,2 tanycytes of WGA and of the proteins clathrin and Rab4 indicates that part of the internalised WGA follows the degradative pathway and part is sorted to a transcytotic pathway and that the transcytotic and the secretory pathways might intersect. Financial support was provided by grants 01/1050, from FIS, Spain (to J.L.B.) and 1030265, from FONDECYT, Chile (to E.M.R.)     

Physiological and biochemical responses to dietary protein in the omnivore passerine Zonotrichia capensis (Emberizidae)

We studied the physiological, biochemical and morphological responses of the omnivore sparrow Zonotrichia capensis, a small opportunistic passerine from Central Chile acclimated to high- and low-protein diets. After 4 weeks of acclimation to 30% (high-protein group) or 7% (low-protein group) dietary casein, we collected urine and plasma for nitrogen waste production and osmometry analysis, and measured gross renal morphology. Plasma osmolality and hematocrit were not significantly affected by dietary treatment, but urine osmolality was higher in the high-protein group than in the low-protein group. Kidney and heart masses were higher in animals acclimated to the high-protein diet. Mean total nitrogen waste was significantly higher in the high-protein group, but the proportions of nitrogen excreted as uric acid, urea and ammonia were unaffected by diet. Our data suggest that the response of Z. capensis to an increase in dietary protein content is through greater amounts of total nitrogen excretion and hypertrophy of kidney structures, without any modification of the proportion of excretory compounds.     

Induced ovulation and egg deposition in the direct developing anuran <Emphasis Type="Italic">Eleutherodactylus coqui</Emphasis>

This study investigates ovulation and egg deposition behaviors in the anuran Eleutherodactylus coqui from Puerto Rico in response to stimulation with gonadotropin and gonadotropin releasing hormones. Five hormones were tested by injection over a range of doses, including mammalian LHRH, avian LHRH, fish LHRH, D-Ala6, des-Gly10 ethylamide LHRH and hCG. We report a low level of ovulation and egg deposition in response to all hormones, with the most complete and consistent results from the non-natural D-Ala6, des-Gly10 ethylamide LHRH derivative. To confirm the viability of eggs produced in this manner we performed in vitro fertilization experiments that resulted in the development of normal frogs. Reproductive behaviors in E. coqui are apparently not controlled by a mammalian form of LHRH as reported in other common laboratory anuran species. D-Ala6, des-Gly10 ethylamide LHRH induces ovulation and deposition of mature and fertilizable eggs in E. coqui.     

Neutralization of enteric coronaviruses with Escherichia coli cells expressing single-chain Fv-autotransporter fusions

We report here that fusions of single-chain antibodies (scFvs) to the autotransporter beta domain of the IgA protease of Neisseria gonorrhoeae are instrumental in locating virus-neutralizing activity on the cell surface of Escherichia coli. E. coli cells displaying scFvs against the transmissible gastroenteritis coronavirus on their surface blocked in vivo the access of the infectious agent to cultured epithelial cells. This result raises prospects for antiviral strategies aimed at hindering the entry into target cells by bacteria that naturally colonize the same intestinal niches.