A redox system for brain targeted estrogen delivery causes chronic body weight decrease in rats

The effects of 2 redox based carriers for brain directed delivery of estradiol (CDS-E2) and ethinyl estradiol (CDS-EE) on body weight were examined in rats. A single dose of CDS-E2 (3 mg/kg) decreased weight gain in castrate rats for at least 24 days. The dose response of weight gain and LH suppression were compared 12 days and 12 to 25 after CDS-E2 and CDS-EE, respectively, in ovariectomized (OVX) rats. Weight decrease was detected at a lower dose and was significant for longer after drug treatment than LH decrease. Both compounds were more potent than equimolar estradiol or estradiol valerate in reducing weight gain. Intact rats also showed decreased weight gain but were less sensitive to CDS-E2 compared to OVX rats. The effects appeared to be estrogen specific as carrier-linked testosterone had no effect on weight. The mechanisms of sustained and potent drug effects on weight are being explored.     

Rotavirus gene structure and function.

Knowledge of the structure and function of the genes and proteins of the rotaviruses has expanded rapidly. Information obtained in the last 5 years has revealed unexpected and unique molecular properties of rotavirus proteins of general interest to virologists, biochemists, and cell biologists. Rotaviruses share some features of replication with reoviruses, yet antigenic and molecular properties of the outer capsid proteins, VP4 (a protein whose cleavage is required for infectivity, possibly by mediating fusion with the cell membrane) and VP7 (a glycoprotein), show more similarities with those of other viruses such as the orthomyxoviruses, paramyxoviruses, and alphaviruses. Rotavirus morphogenesis is a unique process, during which immature subviral particles bud through the membrane of the endoplasmic reticulum (ER). During this process, transiently enveloped particles form, the outer capsid proteins are assembled onto particles, and mature particles accumulate in the lumen of the ER. Two ER-specific viral glycoproteins are involved in virus maturation, and these glycoproteins have been shown to be useful models for studying protein targeting and retention in the ER and for studying mechanisms of virus budding. New ideas and approaches to understanding how each gene functions to replicate and assemble the segmented viral genome have emerged from knowledge of the primary structure of rotavirus genes and their proteins and from knowledge of the properties of domains on individual proteins. Localization of type-specific and cross-reactive neutralizing epitopes on the outer capsid proteins is becoming increasingly useful in dissecting the protective immune response, including evaluation of vaccine trials, with the practical possibility of enhancing the production of new, more effective vaccines. Finally, future analyses with recently characterized immunologic and gene probes and new animal models can be expected to provide a basic understanding of what regulates the primary interactions of these viruses with the gastrointestinal tract and the subsequent responses of infected hosts.     

High affinity binding of divalent cation to actin monomer is much stronger than previously reported

Monomeric actin is known to bind tightly one divalent cation per molecule. We have quantitatively reinvestigated the affinity of actin for Ca++ and Mg++ using the fluorescent Ca++ chelator Quin2 to induce and measure the dissociation of Ca++ from Ca-actin, supporting these studies with measurements using 45Ca. We found that the KD for Ca-actin is actually 1.9 +/- 0.7 nM. Kinetic analysis supported this result and demonstrated a dissociation rate constant (k-) of 0.013 s-1 and an association rate constant (k+) of 6.8 X 10(6)M-1 s-1 for Ca-actin. Competitive binding studies indicated that the binding affinity of actin for Ca++ is 5.4 times that for Mg++, yielding a calculated KD for Mg-actin of about 10 nM. Thus, the tight-binding of divalent cations to actin is 3-4 orders of magnitude stronger than previously thought.     

Human progesterone receptor binding to mouse mammary tumor virus deoxyribonucleic acid: dependence on hormone and nonreceptor nuclear factor(s)

Using crude progesterone receptor preparations from T47D human breast cancer cells, we show by immunoprecipitation assay that receptor specifically and with high affinity recognizes the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). The use of crude preparations minimizes alterations of receptors or loss of associated factors that may occur during purification. Specific binding was obtained at 1:1 molar ratios of receptor to DNA, and HRE sequences are recognized with an affinity at least 3 orders of magnitude greater than nonspecific DNA. We have compared the DNA-binding activities of different forms of progesterone receptors. The unliganded 8S cytosol receptor had low but detectable binding activity for MMTV DNA. Addition of hormone to cytosol produced a small but consistent 2.5-fold increase. In vitro methods of transforming cytosol receptors from an 8S to a 4S species failed to increase DNA-binding further. By contrast, 4S receptors bound by R5020 in whole cells and extracted from nuclei by salt, displayed a substantially higher (average, 11-fold) binding activity than an equal number of unliganded cytosol receptors. The dissociation constants for cytosol and nuclear receptor binding to MMTV DNA were similar (approximately 2 x 10(-9) M). Thus, nuclear receptors possess a higher capacity for binding to specific recognition sequences. These results suggest that hormone or a hormone-dependent mechanism increases the intrinsic DNA-binding activity of receptors independent of receptor transformation from 8S to 4S. Further experiments indicate that a nonreceptor activity in nuclear extracts can increase the sequence-specific DNA-binding activity of cytosol receptors. This activity is present in both T47D cells and receptor-negative MDA-231 cells. We conclude that the higher DNA-binding activity of the nuclear receptor-hormone complex is due in part to receptor interaction with other nuclear proteins or factors. Such interactions may function to maintain receptors in a disaggregated active complex or to stabilize their binding to specific DNA sites.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

Stimulation of Cellular Thymidine Kinases by Human Cytomegalovirus

Thymidine kinase (TK) activity in WI-38 and MRC-5 human fibroblasts was analyzed by discontinuous polyacrylamide gel electrophoresis (disc-PAGE) and discontinuous glycerol gradient electrophoresis (disc-GEP) after subculture or human cytomegalovirus (HCMV) infection. Two peaks of TK activity with different relative fraction-of-migration (R(f)) values were resolved by disc-PAGE or disc-GEP in extracts from log-phase and infected cells. Growing WI-38 cells expressed a slowly migrating (R(f) = 0.14 PAGE, R(f) = 0.4 GEP) peak of TK activity, which was partially inhibited by 1.0 mM dCTP, but which retained little activity at pH 4.5. Growing MRC cells also displayed a slowly migrating peak (R(f) = 0.10 PAGE) with similar properties. Both cell types expressed a faster-migrating TK activity (R(f) = 0.45 PAGE, R(f) = 0.7 GEP) in the growing and resting state that was strongly inhibited by 1 mM dCTP but retained 50% activity at pH 4.5. When either cell type was infected with HCMV, there was a rapid and high-level stimulation of the slowly migrating form of TK and a slight stimulation of the faster-migrating form. Two strains of HCMV (AD169 and Town) failed to produce an electrophoretically distinct virus TK in either cell type after infection. TK enzymes were partially purified by disc-GEP from extracts of log-phase WI-38 or AD169-infected WI-38 cells. Characterization of these enzymes with respect to phosphate donor specificity, pH optima, thermostability, and salt inhibition showed the HCMV-stimulated TKs to be of cellular origin.