Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M _r forms are found in C. psittaci -infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [³²P]-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M _r IncA bands were phosphorylated. A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci -infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti-IncA antibodies into C. psittaci -infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Vaccines with enhanced costimulation maintain high avidity memory CTL

The avidity of Ag-specific CTL is a critical determinant for clearing viral infection and eliminating tumor. Although previous studies have demonstrated that vaccines using enhanced costimulation will enhance the level and avidity of Ag-specific T cells from naive mice, there are conflicting data about the effects of vaccines using enhanced costimulation (vector or dendritic cell based) on the survival of memory T cells. In this study we have first extended previous observations that primary vaccination with a recombinant vaccinia virus (rV-) expressing a model Ag (LacZ) and a triad of T cell costimulatory molecules (B7-1, ICAM-1, and LFA-3 (designated TRICOM)) enhances the level and avidity of T cells from naive vaccinated C57BL/6 (Thy1.2) mice. Adoptive transfer of Thy1.1 memory CD8(+) T cells into naive Thy1.2 C57BL/6 mice was followed by booster vaccinations with a recombinant fowlpox (rF-)-expressing LacZ (rF-LacZ) or booster vaccinations with rF-LacZ/TRICOM. Analysis of levels of beta-galactosidase tetramer-positive T cells and functional assays (IFN-gamma expression and lytic activity) determined that booster vaccinations with rF-LacZ/TRICOM were superior to booster vaccinations with rF-LacZ in terms of both maintenance and enhanced avidity of memory CD8(+) T cells. Antitumor experiments using a self-Ag (carcinoembryonic Ag (CEA) vaccines in CEA transgenic mice bearing CEA-expressing tumors) also demonstrated that the use of booster vaccinations with vaccines bearing enhanced costimulatory capacity had superior antitumor effects. These studies thus have implications in the design of more effective vaccine strategies.     

Identification and analysis of three myristylated vaccinia virus late proteins.

Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or [3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evidence that the open reading frames had been correctly identified and that each protein is myristylated on a glycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggested that A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.     

Global health policies that support the use of banked donor human milk: a human rights issue

Background

The past ten years have witnessed a rising trend in the prevalence and duration of breastfeeding in Italy, but breastfeeding rates increase in an unequal way; they are higher in the North of Italy than in the South. The purpose of this study was to describe the experiences, expectations and beliefs of a sample of mothers, and to identify differences, if any, between the North and the South of Italy.

Methods

The study was conducted in two regions of Italy, Friuli Venezia Giulia in the Northeast and Basilicata in the South. Two hundred and seventy-nine mothers of infants and children 6 to 23 months of age were interviewed using an 85-item questionnaire including closed and open questions on infant feeding experiences and beliefs, sources of information and support, reasons for intended and actual choices and practices, and some demographic and social variables. Face-to-face interviews were conducted between May 2001 and September 2002. Quantitative and qualitative methods were used for data analysis.

Results

The distribution of the mothers by age, education, employment and parity did not differ from that of the general population of the two regions. The reported rates of initiation and duration of breastfeeding were also similar: 95% started breastfeeding, exclusive breastfeeding was 32% at three and 9% at six months, with 64% and 35% of any breastfeeding, respectively. Some differences were reported in the rates of full breastfeeding, reflecting different ages of introduction of non-nutritive fluids. These, as well as nutritive fluids – including infant formula – and complementary foods, were introduced far too early. Advice on infant feeding was generally provided by health professionals and often was not based on up-to-date recommendations. Mothers were generally aware of the advantages of breastfeeding, but at the same time reported problems that they were not able to solve alone or through social and health system support. Most mothers would welcome the support of a peer counsellor. More mothers in Basilicata than in Friuli Venezia Giulia reported difficulties with breastfeeding related to returning to work and were not familiar with their rights on breastfeeding and maternity leave.

Conclusion

Programmes for the protection, promotion and support of breastfeeding in these and similar regions of Italy should concentrate on better training of health professionals with regards to lactation management, communication, and counselling skills. The addition of trained peer counsellors could reinforce the work done by the health system and, through community involvement, could help change social prejudice in the mid- and long-term. The differences between regions should be taken into account in formulating these programmes to avoid increasing, and possibly to decrease, the current gaps.