SNAP-25 Palmitoylation and Plasma Membrane Targeting Require a Functional Secretory Pathway

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.

Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

Retrograde Transport from the Pre-Golgi Intermediate Compartment and the Golgi Complex Is Affected by the Vacuolar H+-ATPase Inhibitor Bafilomycin A1

The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive ∼80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of β-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER–Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes

Na+/Cl−-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [35S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na+/Cl−-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.

Efficient pyrophosphorolysis by a hepatitis B virus polymerase may be a primer-unblocking mechanism

Effective antiviral agents are thought to inhibit hepatitis B virus (HBV) DNA synthesis irreversibly by chain termination because reverse transcriptases (RT) lack an exonucleolytic activity that can remove incorporated nucleotides. However, since the parameters governing this inhibition are poorly defined, fully delineating the catalytic mechanism of the HBV-RT promises to facilitate the development of antiviral drugs for treating chronic HBV infection. To this end, pyrophosphorolysis and pyrophosphate exchange, two nonhydrolytic RT activities that result in the removal of newly incorporated nucleotides, were characterized by using endogenous avian HBV replication complexes assembled in vivo. Although these activities are presumed to be physiologically irrelevant for every polymerase examined, the efficiency with which they are catalyzed by the avian HBV-RT strongly suggests that it is the first known polymerase to catalyze these reactions under replicative conditions. The ability to remove newly incorporated nucleotides during replication has important biological and clinical implications: these activities may serve a primer-unblocking function in vivo. Analysis of pyrophosphorolysis on chain-terminated DNA revealed that the potent anti-HBV drug β-l-(−)-2′,3′-dideoxy-3′-thiacytidine (3TC) was difficult to remove by pyrophosphorolysis, in contrast to ineffective chain terminators such as ddC. This disparity may account for the strong antiviral efficacy of 3TC versus that of ddC. The HBV-RT pyrophosphorolytic activity may therefore be a novel determinant of antiviral drug efficacy, and could serve as a target for future antiviral drug therapy. The strong inhibitory effect of cytoplasmic pyrophosphate concentrations on viral DNA synthesis may also partly account for the apparent slow rate of HBV genome replication.

Magnetars

Recent x-ray observations have shown that a substantial fraction of newly born neutron stars have magnetic fields of several 1014 G. They reveal themselves as soft gamma repeaters and anomalous x-ray pulsars and may account for the missing radio pulsars in young supernova remnants.

Human cytomegalovirus inhibits antigen presentation by a sequential multistep process.

The human cytomegalovirus (HCMV) genomic unique short (US) region encodes a family of homologous genes essential for the inhibition of major histocompatibility complex (MHC) class I-mediated antigen presentation during viral infection. Here we show that US3, the only immediate early (IE) gene within the US region, encodes an endoplasmic reticulum-resident glycoprotein that prevents intracellular transport of MHC class I molecules. In contrast to the rapid degradation of newly synthesized MHC class I heavy chains mediated by the early gene product US11, we found that US3 retains stable MHC class I heterodimers in the endoplasmic reticulum that are loaded with peptides while retained in the ER. Consistent with the expression pattern of US3 and US11, MHC class I molecules are retained but not degraded during the IE period of infection. Our data identify the first nonregulatory role of an IE protein of HCMV and suggest that HCMV uses different T-cell escape strategies at different times during the infectious cycle.

Gene recognition via spliced sequence alignment.

Gene recognition is one of the most important problems in computational molecular biology. Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored. Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes. This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons. On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99%. The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing. Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic. The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.

Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains.

Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally.

A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

Haploidy and androgenesis in Drosophila.

Adrogenesis, development from paternal but not maternal chromosomes, can be induced to occur in some organisms, including vertebrates, but has only been reported to occur naturally in interspecific hybrids of the Sicilian stick insect. Androgenesis has not been described previously in Drosophila. We now report the recovery of androgenetic offspring from Drosophila melanogaster females mutant for a gene that affects an oocyte- and embryo-specific alpha-tubulin. The androgenetic exceptions are X,X diploid females that develop from haploid embryos and express paternal markers on all 4 chromosomes. The exceptional females arise by fusion of haploid cleavage nuclei or failure of newly replicated haploid chromosomes to segregate, rather than fusion of two inseminating sperm. The frequency of androgenetic offspring is greatly enhanced by a partial loss-of-function mutant of the NCD (nonclaret disjunctional) microtubule motor protein, suggesting that wild-type NCD functions is pronuclear fusion. Diploidization of haploid paternal chromosome complements results in complete genetic homozygosity, which could facilitate studies of gene variation and mutational load in populations.

Worldwide violence against women legislation: an equity approach

OBJECTIVES: To describe the recommendations and interventions addressing violence against women (VAW) in vulnerable women (disabled, pregnant, ethnic minority, immigrant and older women) in key documents and laws enacted in different countries. METHODS: Content analysis of key documents for the development of VAW policies and laws: The United Nations Handbook for Legislation on Violence Against Women Advance Version, the Model of Laws and Policies on Intrafamiliar Violence Against Women of the Pan-American Health Organization and Recommendation No. R(2002)5 of the Committee of Ministers of the European Council. The content of the 62 VAW laws was also analyzed. RESULTS: Key documents demonstrate the importance of eliminating any obstacle facing disabled, pregnant, immigrant, ethnic minority or older women when accessing VAW services. Only 12 laws mention one or more of these groups of vulnerable women. Pregnant, disabled and ethnic minority women are the groups most often mentioned. In these laws, references to punitive measures, action plans and specific strategies to guarantee access to VAW resources are the most common interventions. CONCLUSION: Decisive interventions addressing the specific needs of disabled, pregnant, immigrant, ethnic minority and older women are needed in order to achieve a broader equity approach in VAW legislation.

Violencia de género en mujeres inmigrantes y españolas: magnitud, respuestas ante el problema y políticas existentes

Objetivo: Comparar la prevalencia de la violencia de género entre mujeres inmigrantes y españolas. Describir sus respuestas ante esta situación y posibles diferencias entre ellas. Identificar intervenciones ya existentes en España sobre prevención y atención sociosanitaria de violencia de género dirigidas a inmigrantes. Métodos: Estudio transversal mediante encuesta autoadministrada en 10.202 mujeres que acudieron a centros de atención primaria en España (2006-2007). Análisis de contenido del informe de seguimiento de la ley 1/2004 de medidas de protección integral contra la violencia de género remitido por las comunidades autónomas (CC.AA.) (2005) y las leyes y planes autonómicos más recientes. Resultados: La prevalencia de violencia de género en las españolas es del 14,3% y en las inmigrantes del 27,3%. La probabilidad de violencia de género en las inmigrantes es mayor (odds ratio ajustada: 2,06; intervalo de confianza del 95%: 1,61–2,64). Las inmigrantes dijeron haber denunciado a su pareja con más frecuencia, así como que no sabían resolver su situación. Algunas CC.AA. ya han emprendido intervenciones para superar las barreras de acceso a los servicios sociosanitarios, pero sólo tres facilitan el número de mujeres inmigrantes beneficiarias de ayudas económicas y laborales hasta 2005. Conclusiones: Existe una desigual distribución en la prevalencia de la violencia de género según el país de origen, afectando en mayor medida a las mujeres inmigrantes. Éstas denuncian con más frecuencia que las españolas, pero tal actuación no supone una garantía de resultados efectivos. Aunque se han identificado otras intervenciones específicas en algunas CC.AA., sería necesario evaluarlas para asegurar que las mujeres inmigrantes se están beneficiando.

Social and immigration factors in intimate partner violence among Ecuadorians, Moroccans and Romanians living in Spain

Background: Intimate partner violence (IPV) against women occurs in all countries, all cultures and at every level of society; however, some populations may be at greater risk than others. The aim of this study was to explore IPV prevalence among Ecuadorian, Moroccan and Romanian immigrant women living in Spain and its possible association with their personal, family, social support and immigration status characteristics. Methods: Cross-sectional study of 1607 adult immigrant women residing in Barcelona, Madrid and Valencia (2011). Prevalence rates and adjusted odds ratios (AORs) were calculated, with current IPV being the outcome. Different women’s personal (demographic), family, social support and immigration status characteristics were considered as explicative and control variables. All analyses were separated by women’s country of origin. Results: Current IPV prevalence was 15.57% in Ecuadorians, 10.91% in Moroccans and 8.58% in Romanians. Some common IPV factors were found, such as being separated and/or divorced. In Romanians, IPV was also associated with lack of social support [AOR 5.96 (1.39–25.62)] and low religious involvement [AOR 2.17 (1.06–4.43)]. The likelihood of current IPV was lower among women without children or other dependants in this subgroup [AOR 0.29 (0.093–0.92)]. Conclusion: The IPV prevalence rates obtained for Moroccan, Romanian and Ecuadorian women residing in Spain were similar. Whereas the likelihood of IPV appeared to be relatively evenly distributed among Moroccan and Ecuadorian women, it was higher among Romanian women in socially vulnerable situations related to family responsibilities and the lack of support networks. The importance of intervention in the process of separation and divorce was common to all women.

Racism, Other Discriminations and Effects on Health

We study the probability of perceived racism/other forms of discrimination on immigrant and Spanish populations within different public spheres and show their effect on the health of immigrants using a cross-sectional design (ENS-06). Variables: perceived racism/other forms of discrimination (exposure), socio-demographic (explicative), health indicators (dependent). Frequencies, prevalences, and bivariate/multivariate analysis were conducted separately for men (M) and women (W). We estimated the health problems attributable to racism through the population attributable proportion (PAP). Immigrants perceived more racism than Spaniards in workplace (ORM = 48.1; 95 % CI 28.2–82.2), and receiving health care (ORW = 48.3; 95 % CI 24.7–94.4). Racism and other forms of discrimination were associated with poor mental health (ORM = 5.6; 95 % CI 3.9–8.2; ORW = 7.3; 95 % CI 4.1–13.0) and injury (ORW = 30.6; 95 % CI 13.6–68.7). It is attributed to perceived racism the 80.1 % of consumption of psychotropics (M), and to racism with other forms of discrimination the 52.3 % of cases of injury (W). Racism plays a role as a health determinant.