Regulation of T cell dependent immune responses by TIM family members

The T cell immunoglobulin mucin (TIM) proteins are type I membrane glycoproteins expressed on T cells and containing common structural motifs. While our understanding on the distribution and functions of TIM family members is still incomplete, data from several recent reports indicate that these proteins, together with T cell receptor and costimulatory signals, regulate the expansion and effector functions of T helper cells. In the current review, we provide evidences indicating that TIM-3 is capable of modulating the function of CD4(+)CD25(+) regulatory T cells and inhibiting aggressive Th1 mediated auto- and allo-immune responses. Similarly, additional data suggest that TIM-2 molecules function by negatively regulating Th2 immune responses. In contrast, TIM-1 appears to be an activation molecule for all T cells, although the mechanisms through which TIM-1 activates T cells remain to be elicited.     

Viral Glycoproteins Accumulate in Newly Formed Annulate Lamellae following Infection of Lymphoid Cells by Human Herpesvirus 6

Ultrastructural analysis of HSB-2 T-lymphoid cells and human cord blood mononuclear cells infected with human herpesvirus 6 revealed the presence, in the cell cytoplasm, of annulate lamellae (AL), which were absent in uninfected cells. Time course analysis of the appearance of AL following viral infection showed that no AL were visible within the first 72 h postinfection and that their formation correlated with the expression of the late viral glycoprotein gp116. The requirement of active viral replication for AL neoformation was further confirmed by experiments using inactivated virus or performed in presence of the viral DNA polymerase inhibitor phosphonoacetic acid. Both conventional electron microscopic examination and immunogold fracture labeling with anti-endoplasmic reticulum antibodies indicated a close relationship of AL with the endoplasmic reticulum and nuclear membranes. However, when the freeze-fractured cells were immunogold labeled with an anti-gp116 monoclonal antibody, AL membranes were densely labeled, whereas nuclear membranes and endoplasmic reticulum cisternae appeared virtually unlabeled, showing that viral envelope glycoproteins selectively accumulate in AL. In addition, gold labeling with Helix pomatia lectin and wheat germ agglutinin indicated that AL cisternae, similar to cis-Golgi membranes, contain intermediate, but not terminal, forms of glycoconjugates. Taken together, these results suggest that in this cell-virus system, AL function as a viral glycoprotein storage compartment and as a putative site of O-glycosylation.     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.     

Causes of Stranding and Mortality,and Final Disposition of Loggerhead Sea Turtles (Caretta caretta) Admitted to a Wildlife Rehabilitation Center in Gran Canaria Island,Spain (1998-2014): A Long-Term Retrospective Study

Aims

The aims of this study were to analyze the causes of stranding of 1,860 loggerhead turtles (Caretta caretta) admitted at the Tafira Wildlife Rehabilitation Center in Gran Canaria Island, Spain, from 1998 to 2014, and to analyze the outcomes of the rehabilitation process to allow meaningful auditing of its quality.

Methods

Primary causes of morbidity were classified into seven categories: entanglement in fishing gear and/or plastics, ingestion of hooks and monofilament lines, trauma, infectious disease, crude oil, other causes, and unknown/undetermined. Final dispositions were calculated as euthanasia (E_r), unassisted mortality (M_r), and release (R_r) rates. Time to death (T_d) for euthanized and dead turtles, and length of stay for released (T_r) turtles were evaluated.

Results

The most frequent causes of morbidity were entanglement in fishing gear and/or plastics (50.81%), unknown/undetermined (20.37%), and ingestion of hooks (11.88%). The final disposition of the 1,634 loggerhead turtles admitted alive were: E_r = 3.37%, M_r = 10.34%, and R_r = 86.29%. E_r was significantly higher in the trauma category (18.67%) compared to the other causes of admission. The highest M_r was observed for turtles admitted due to trauma (30.67%). The highest R_r was observed in the crude oil (93.87%) and entanglement (92.38%) categories. The median T_r ranged from 12 days (unknown) to 70 days (trauma).

Conclusions

This survey is the first large-scale epidemiological study on causes of stranding and mortality of Eastern Atlantic loggerheads and demonstrates that at least 71.72% of turtles stranded due to anthropogenic causes. The high R_r (86.29%) emphasizes the importance of marine rehabilitation centers for conservation purposes. The stratified analysis by causes of admission of the three final disposition rates, and the parameters T_d and T_r should be included in the outcome research of the rehabilitation process of sea turtles in order to allow comparative studies between marine rehabilitation centers around the world.     

Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.     

Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels

This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.     

Microorganisms under high pressure — Adaptation,growth and biotechnological potential

Hydrostatic pressure is a well-known physical parameter which is now considered an important variable of life, since organisms have the ability to adapt to pressure changes, by the development of resistance against this variable. In the past decades a huge interest in high hydrostatic pressure (HHP) technology is increasingly emerging among food and biosciences researchers. Microbial specific stress responses to HHP are currently being investigated, through the evaluation of pressure effects on biomolecules, cell structure, metabolic behavior, growth and viability. The knowledge development in this field allows a better comprehension of pressure resistance mechanisms acquired at sub-lethal pressures. In addition, new applications of HHP could arise from these studies, particularly in what concerns to biotechnology. For instance, the modulation of microbial metabolic pathways, as a response to different pressure conditions, may lead to the production of novel compounds with potential biotechnological and industrial applications. Considering pressure as an extreme life condition, this review intends to present the main findings so far reported in the scientific literature, focusing on microorganisms with the ability to withstand and to grow in high pressure conditions, whether they have innated or acquired resistance, and show the potential of the application of HHP technology for microbial biotechnology.