Human and viral Golgi anti-apoptotic proteins (GAAPs) oligomerize via different mechanisms and monomeric GAAP inhibits apoptosis and modulates calcium.

Golgi anti-apoptotic proteins (GAAPs) are hydrophobic proteins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca(2+) content of intracellular stores, and regulate Ca(2+) fluxes. GAAP was discovered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca(2+)-modulating membrane proteins. Most TMBIM members show multiple bands when analyzed by SDS-PAGE, suggesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligomerization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligomerization of hGAAP is independent of cysteines, but oligomerization of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellular Ca(2+) stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.

Analysis of the anti-apoptotic activity of four vaccinia virus proteins demonstrates that B13 is the most potent inhibitor in isolation and during viral infection.

Vaccinia virus (VACV) is a large dsDNA virus encoding ~200 proteins, several of which inhibit apoptosis. Here, a comparative study of anti-apoptotic proteins N1, F1, B13 and Golgi anti-apoptotic protein (GAAP) in isolation and during viral infection is presented. VACVs strains engineered to lack each gene separately still blocked apoptosis to some degree because of functional redundancy provided by the other anti-apoptotic proteins. To overcome this redundancy, we inserted each gene separately into a VACV strain (vv811) that lacked all these anti-apoptotic proteins and that induced apoptosis efficiently during infection. Each protein was also expressed in cells using lentivirus vectors. In isolation, each VACV protein showed anti-apoptotic activity in response to specific stimuli, as measured by immunoblotting for cleaved poly(ADP ribose) polymerase-1 and caspase-3 activation. Of the proteins tested, B13 was the most potent inhibitor, blocking both intrinsic and extrinsic stimuli, whilst the activity of the other proteins was largely restricted to inhibition of intrinsic stimuli. In addition, B13 and F1 were effective blockers of apoptosis induced by vv811 infection. Finally, whilst differences in induction of apoptosis were barely detectable during infection with VACV strain Western Reserve compared with derivative viruses lacking individual anti-apoptotic genes, several of these proteins reduced activation of caspase-3 during infection by vv811 strains expressing these proteins. These results illustrated that vv811 was a useful tool to determine the role of VACV proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 was the most potent.

Six-transmembrane topology for Golgi anti-apoptotic protein (GAAP) and Bax inhibitor 1 (BI-1) provides model for the transmembrane Bax inhibitor-containing motif (TMBIM) family.

The Golgi anti-apoptotic protein (GAAP) is a hydrophobic Golgi protein that regulates intracellular calcium fluxes and apoptosis. GAAP is highly conserved throughout eukaryotes and some strains of vaccinia virus (VACV) and camelpox virus. Based on sequence, phylogeny, and hydrophobicity, GAAPs were classified within the transmembrane Bax inhibitor-containing motif (TMBIM) family. TMBIM members are anti-apoptotic and were predicted to have seven-transmembrane domains (TMDs). However, topology prediction programs are inconsistent and predicted that GAAP and other TMBIM members have six or seven TMDs. To address this discrepancy, we mapped the transmembrane topology of viral (vGAAP) and human (hGAAP), as well as Bax inhibitor (BI-1). Data presented show a six-, not seven-, transmembrane topology for vGAAP with a putative reentrant loop at the C terminus and both termini located in the cytosol. We find that this topology is also conserved in hGAAP and BI-1. This places the charged C terminus in the cytosol, and mutation of these charged residues in hGAAP ablated its anti-apoptotic function. Given the highly conserved hydrophobicity profile within the TMBIM family and recent phylogenetic data indicating that a GAAP-like protein may have been the ancestral progenitor of a subset of the TMBIM family, we propose that this vGAAP topology may be used as a model for the remainder of the TMBIM family of proteins. The topology described provides valuable information on the structure and function of an important but poorly understood family of proteins.

Implementation of High Intensity Interval Training and Autoregulatory Progressive Resistance Exercise in a Law Enforcement Training Academy.

The purpose of this investigation was to assess the feasibility and efficacy of implementing autoregulatory progressive resistance exercise (APRE) and high intensity interval training (HIIT) methodologies to improve physical fitness and occupational physical ability in police cadets. Two law enforcement academy classes were stratified into a standard care academy training cohort (SC; n=32, m=27, f=5) and a high performance cohort (HP; n=31; m=27, f=4) that utilized APRE and HIIT methodologies during a 17-week academy training program. Demographic, internal loading parameters, anthropometric, fitness outcomes (i.e., 1.5-mile run, 1-repetition maximum bench press, sit-up repetitions, push-up repetitions, & 300m run) and timed completion of a occupation physical ability test (OPAT) were collected at three academy time points (entrance, mid-point and exit). Mixed factor (time vs. group) repeated measures ANOVA were used to evaluate the effects of the training intervention on performance outcomes. Significance was set at p<0.05. Both groups demonstrated significant improvements in all fitness outcomes except the OPAT from entrance to exit tests (p<0.05). The HP experienced greater improvements in push-up performance compared to the SC (p<0.001). OPAT time decreased in both groups from entrance to midpoint, but significantly increased from baseline to exit (p<.05). Despite similar inter-group fitness improvements, the HP reported lower session RPE values (p<0.01), indicating fitness adaptations occurred at a lower internal load. This study demonstrated the feasibility of successfully implementing APRE and HIIT methodologies within a cadet population. Furthermore, these methodologies produced similar improvements in cadet fitness and occupational performance at a lower internal load.

Serological responses in humans to the smallpox vaccine LC16m8.

In response to potential bioterrorism with smallpox, members of the Japanese Self-Defense Forces were vaccinated with vaccinia virus (VACV) strain LC16m8, an attenuated smallpox vaccine derived from VACV strain Lister. The serological response induced by LC16m8 to four virion-surface proteins and the intracellular mature virus (IMV) and extracellular enveloped virus (EEV) was investigated. LC16m8 induced antibody response against the IMV protein A27 and the EEV protein A56. LC16m8 also induced IMV-neutralizing antibodies, but unlike the VACV strain Lister, did not induce either EEV-neutralizing antibody or antibody to EEV protein B5, except after revaccination. Given that B5 is the only target for EEV-neutralizing antibody and that neutralization of both IMV and EEV give optimal protection against orthopoxvirus challenge, these data suggest that immunity induced by LC16m8 might be less potent than that deriving from strain Lister. This potential disadvantage should be balanced against the advantage of the greater safety of LC16m8.

Kinematic analysis of the powerlifting style squat and the conventional deadlift during competition: is there a cross-over effect between lifts?

Many individuals involved in the sport of powerlifting believe that the squat and deadlift have such similar lifting characteristics that the lifts yield comparable training results. The aim of this study was to compare and contrast biomechanical parameters between the conventional style deadlift and the back squat performed by 25 lifters competing in regional powerlifting championship. The 3-dimensional analysis incorporated 4 60 Hz synchronized video cameras for collecting data from 25 participants. Parameters were quantified at the sticking point specific to each lift. Kinematic variables were calculated at the hip, knee, and ankle. Paired (samples) t-tests were used to detect significant differences in the kinematic mean scores for the different lift types. The statistical analysis revealed significant differences exist between the squat (0.09 m/s) and the deadlift (0.20 m/s) vertical bar velocities. Differences were found for angular position of the hip, knee, and ankle between lifts. The sticking point thigh angles were quantified as 32.54 +/- 3.02 and 57.42 +/- 4.57 for the squat and deadlift, respectively. Trunk angles were 40.58 +/- 6.29 (squat) and 58.30 +/- 7.15 (deadlift). The results indicate the back squat represents a synergistic or simultaneous movement, whereas the deadlift demonstrates a sequential or segmented movement. The kinematic analysis of the squat and the conventional deadlift indicate that the individual lifts are markedly different (p < 0.01), implying that no direct or specific cross-over effect exists between the individual lifts.

The effect of forefoot varus on postural stability.

STUDY DESIGN: Counterbalanced experimental design study comparing a group of subjects with greater than or equal to 70 of forefoot varus (MFV) to a group with less than 70 of forefoot varus (LFV). OBJECTIVES: To investigate the effect of forefoot varus on single-limb stance postural stability (PS). BACKGROUND: Impaired PS has been implicated as a potential risk factor for sustaining acute foot and ankle injuries. The identification of variables that deleteriously affect PS may be important in the prevention of future injuries. METHODS AND MEASURES: Postural stability of the MFV group (n = 20) and the LFV group (n = 12) was assessed during right and left single-limb stance and eyes-open and eyes-closed conditions. Standard deviations of the x-axis and y-axis ground reaction forces measured via a force platform were used to represent anteroposterior (AP) and mediolateral (ML) PS, respectively. The mean of 3 successful 5-second trials of each testing condition was calculated and used for subsequent data analysis using 3-way mixed-model ANOVAs with 1 between-subject and 2 within-subject factors. RESULTS: The AP PS scores of the MFV group were significantly greater than those of the LFV group (P < .05). ML PS scores, although higher in the MFV group, were not significantly different from those of the LFV group. Both groups had significantly greater AP and ML PS scores during the eyes-closed versus the eyes-open condition (P < .05). CONCLUSIONS: The results suggest that the presence of greater than or equal to 70 of forefoot varus may significantly impair AP PS. The decreased stability associated with increased forefoot varus may be due to decreased joint congruity and consequently an increased reliance on soft tissue structures for stability.

hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2.

Golgi antiapoptotic proteins (GAAPs) are highly conserved Golgi membrane proteins that inhibit apoptosis and promote Ca(2+) release from intracellular stores. Given the role of Ca(2+) in controlling cell adhesion and motility, we hypothesized that human GAAP (hGAAP) might influence these events. In this paper, we present evidence that hGAAP increased cell adhesion, spreading, and migration in a manner that depended on the C-terminal domain of hGAAP. We show that hGAAP increased store-operated Ca(2+) entry and thereby the activity of calpain at newly forming protrusions. These hGAAP-dependent effects regulated focal adhesion dynamics and cell migration. Indeed, inhibition or knockdown of calpain 2 abrogated the effects of hGAAP on cell spreading and migration. Our data reveal that hGAAP is a novel regulator of focal adhesion dynamics, cell adhesion, and migration by controlling localized Ca(2+)-dependent activation of calpain.

Phase I trial of recombinant modified vaccinia ankara encoding Epstein-Barr viral tumor antigens in nasopharyngeal carcinoma patients.

Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma, a high incidence tumor in Chinese populations, in which tumor cells express the two EBV antigens EB nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2). Here, we report the phase I trial of a recombinant vaccinia virus, MVA-EL, which encodes an EBNA1/LMP2 fusion protein designed to boost T-cell immunity to these antigens. The vaccine was delivered to Hong Kong patients with nasopharyngeal carcinoma to determine a safe and immunogenic dose. The patients, all in remission more than 12 weeks after primary therapy, received three intradermal MVA-EL vaccinations at three weekly intervals, using five escalating dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming unit (pfu). Blood samples were taken during prescreening, immediately before vaccination, one week afterward and at intervals up to one year later. Immunogenicity was tested by IFN-γ ELIspot assays using complete EBNA1 and LMP2 15-mer peptide mixes and known epitope peptides relevant to patient MHC type. Eighteen patients were treated, three per dose level one to four and six at the highest dose, without dose-limiting toxicity. T-cell responses to one or both vaccine antigens were increased in 15 of 18 patients and, in many cases, were mapped to known CD4 and CD8 epitopes in EBNA1 and/or LMP2. The range of these responses suggested a direct relationship with vaccine dose, with all six patients at the highest dose level giving strong EBNA1/LMP2 responses. We concluded that MVA-EL is both safe and immunogenic, allowing the highest dose to be forwarded to phase II studies examining clinical benefit.

Fatal cases of influenza A(H3N2) in children: insights from whole genome sequence analysis.

During the Northern Hemisphere winter of 2003-2004 the emergence of a novel influenza antigenic variant, A/Fujian/411/2002-like(H3N2), was associated with an unusually high number of fatalities in children. Seventeen fatal cases in the UK were laboratory confirmed for Fujian/411-like viruses. To look for phylogenetic patterns and genetic markers that might be associated with increased virulence, sequencing and phylogenetic analysis of the whole genomes of 63 viruses isolated from fatal cases and non fatal "control" cases was undertaken. The analysis revealed the circulation of two main genetic groups, I and II, both of which contained viruses from fatal cases. No associated amino acid substitutions could be linked with an exclusive or higher occurrence in fatal cases. The Fujian/411-like viruses in genetic groups I and II completely displaced other A(H3N2) viruses, but they disappeared after 2004. This study shows that two A(H3N2) virus genotypes circulated exclusively during the winter of 2003-2004 in the UK and caused an unusually high number of deaths in children. Host factors related to immune state and differences in genetic background between patients may also play important roles in determining the outcome of an influenza infection.

Fatal cases of influenza a in childhood.

BACKGROUND: In the northern hemisphere winter of 2003-04 antigenic variant strains (A/Fujian/411/02 -like) of influenza A H3N2 emerged. Circulation of these strains in the UK was accompanied by an unusually high number of laboratory confirmed influenza associated fatalities in children. This study was carried out to better understand risk factors associated with fatal cases of influenza in children. METHODOLOGY/PRINCIPAL FINDINGS: Case histories, autopsy reports and death registration certificates for seventeen fatal cases of laboratory confirmed influenza in children were analyzed. None had a recognized pre-existing risk factor for severe influenza and none had been vaccinated. Three cases had evidence of significant bacterial co-infection. Influenza strains recovered from fatal cases were antigenically similar to those circulating in the community. A comparison of protective antibody titres in age stratified cohort sera taken before and after winter 2003-04 showed that young children had the highest attack rate during this season (21% difference, 95% confidence interval from 0.09 to 0.33, p = 0.0009). Clinical incidences of influenza-like illness (ILI) in young age groups were shown to be highest only in the years when novel antigenic drift variants emerged. CONCLUSIONS/SIGNIFICANCE: This work presents a rare insight into fatal influenza H3N2 in healthy children. It confirms that circulating seasonal influenza A H3N2 strains can cause severe disease and death in children in the apparent absence of associated bacterial infection or predisposing risk factors. This adds to the body of evidence demonstrating the burden of severe illness due to seasonal influenza A in childhood.

Vascular endothelial growth factor and immunosuppression in cancer: current knowledge and potential for new therapy.

Two decades of research into the role of immunosuppression and angiogenesis in tumor biology have revealed multiple links between the two. Vascular endothelial growth factor, originally thought to be solely involved in vascular growth and permeability, has emerged as a significant agent of immune tolerance in the tumor microenvironment. This review examines two major elements of this field: the research behind the role of vascular endothelial growth factor in immunosuppression, especially as pertains to dendritic cell function; and the subsequent research into the potential for using antiangiogenic therapy to both starve tumors by hypoxia and enhance the response of tumors to immunotherapy. Several strategies tested so far have yielded incomplete, yet promising, results.