Cutting edge: CD8+ T cell priming in the absence of NK cells leads to enhanced memory responses

It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8(+) T cell fate by killing recently activated CD8(+) T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8(+) T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8(+) T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8(+) T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.     

Systematic analysis of the amino acid residues of human papillomavirus type 16 E7 conserved region 3 involved in dimerization and transformation

The human papillomavirus (HPV) E7 oncoprotein exists as a dimer and acts by binding to many cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. Dimerization of E7 is attributed primarily to the C-terminal domain, referred to as conserved region 3 (CR3). CR3 is highly structured and is necessary for E7's transformation ability. It is also required for binding of numerous E7 cellular targets. To systematically analyze the molecular mechanisms by which HPV16 E7 CR3 contributes to carcinogenesis, we created a comprehensive panel of mutations in residues predicted to be exposed on the surface of CR3. We analyzed our novel collection of mutants, as well as mutants targeting predicted hydrophobic core residues of the dimer, for the ability to dimerize. The same set of mutants was also assessed functionally for transformation capability in a baby rat kidney cell assay in conjugation with activated ras. We show that some mutants of HPV16 E7 CR3 failed to dimerize yet were still able to transform baby rat kidney cells. Our results identify several novel E7 mutants that abrogate transformation and also indicate that E7 does not need to exist as a stable dimer in order to transform cells.     

Chemical Composition and Interpopulation Variability of Essential Oils of Taxus baccata L. from Serbia

The composition of the essential oil of the twigs and needles of Taxus baccata L. from three natural populations in Serbia has been determined by GC/MS analysis. Of the 91 detected compounds, 87 were identified. The most abundant compound classes were aliphatic alcohols, terpenes, aliphatic hydrocarbons, and aliphatic aldehydes, which together comprised ca. 86.92% of the total oil composition. The dominant constituents were oct‐1‐en‐3‐ol (23.48%), (3Z)‐hex‐3‐en‐1‐ol (11.46%; aliphatic alcohols), and myrtenol (11.38%; oxygenated monoterpene). The PCA of 22 selected compounds revealed differentiations of populations based on geographic distribution. The CA showed that Populations I and II from the Dinaric Alps were similar, and that Population III from the Balkan mountain system was distinct. This was the first investigation of interpopulation variability of T. baccata populations based on essential oil composition. The results of this study were compared with those of previous studies concerning volatile compounds produced by Taxus species. The results indicate that the essential oil content of T. baccata populations from this study is unique, mostly resembling the population from southeast Serbia.     

The Phenolics of the Ornithogalum umbellatum L. (Hyacinthaceae): Phytochemical and Ecological Characterization

Ornithogalum umbellatum L. is a widely distributed species in Europe, that exhibits considerable variability at ecological, morphological, anatomical, and karyological level. Previous reports of the chemical investigations among Ornithogalum species indicate significant diversity of the secondary metabolites, as well. Knowing that environment affects the phenolic composition in plants to a large extent, the main objective of the research was to define relationship between phytochemical and ecological characters. To estimate an environmental influence on these results, plant material was collected at four habitats that differ in ecological factors and belong to two biogeographical regions: the Balkan Peninsula and the Pannonian Plane. Measured phytochemical characters are yield of dry extract, total phenolic and flavonoid contents, the presence of selected phenolic compounds as well as free‐radical scavenging activity (neutralization of DPPH and OH radicals). Results revealed that all analyzed phytochemical parameters differ between investigated O. umbellatum samples. The moisture level of habitat has the highest correlation, either positive or negative, with most of phytochemical characters, and is followed by temperature and soil reaction. Light intensity and nitrogen level have mostly moderate correlation coefficient with phytochemical characters. More complex correlation is revealed between ecological factors and nine phenolic compounds, with three observed patterns of relationship.     

Ancient split of major genetic lineages of European Black Pine: evidence from chloroplast <Emphasis>DNA</Emphasis>

The European Black Pine (Pinus nigra Arn.) has a long and complex history. Genetic distance and frequency analyses identified three differentiated genetic groups, which corresponded to three wide geographical areas: Westerns Mediterranean, Balkan Peninsula and Asia Minor. These groups shared common ancestors (14.75 and 10.72 Ma). The most recent splits occurred after the Messinian Salinity Crisis (4.37 Ma) and the Early–Middle Pleistocene Transitions (0.93 Ma). The posterior ancestral population size (Na) is 260,000–265,000 individuals. Each pool is further fragmented, with evidence of a phylogeographic structure (N _st  > G _st ) typically observed in some natural populations from the Western Mediterranean region and the Balkan Peninsula. The laboratory analysis was performed by fragment analysis—i.e. electrophoretic sizing of polymerase chain reaction fragments, combined with the sequencing analysis of 33 % of all individuals as a control. Intense sampling of chloroplast DNA polymorphisms (3154 individuals and 13 markers: SNPs and SSRs) over the full area of the species’ natural distribution indicated moderate among-population variability (G _st(nc) ≤ 0.177) in various parts of its range. These results indicate that the natural populations have long migration histories that differ from one another and that they have been strongly phylogeographically affected by complex patterns of isolation, speciation and fragmentation. Long and varying climatic fluctuations in the region of the principal genetic group have been the probable cause of different forest community associations with different successional patterns resulting in interglacial refugia vs. macro long-term refugia.     

Heterogeneity of the hemoglobin of the Ohrid trout (Salmo L. typicus)

We have analyzed the hemoglobins of five individual trout from the Ohrid Lake (Salmo L. typicus) by electrophoretic methods, by reversed-phase high-performance liquid chromatography, and by limited structural analyses. The two major classes of hemoglobin are type I (35% of total) and type IV (65%). Type IV is the major oxygen-transporting hemoglobin; it consists of three types of chain (in about equal quantities) and three types of chain (one major and two minor types). Several structural differences have been observed between these three (IV) chains and between the three (IV) chains, suggesting a complex genetic system governing the synthesis of these proteins. Moreover, a few amino acid substitutions occur at positions involved in contacts between chains, which suggests that differences in oxygen affinity may exist between these various type IV hemoglobins. Type I hemoglobin is less complex because it contains one type of chain and two chains; the latter two differ in numerous positions, suggesting duplications of the (I)-globin gene. The and chains of type I hemoglobin differ considerably from the and chains of type IV hemoglobin, indicating the existence of (I)- and (I)-globin genes separate from the (IV)- and (IV)- globin genes.This study was supported in part by the Yugoslav-American Joint Funds, pp 812 (to G.D.E.), and by United States Public Health Service Research Grant HLB-05168 (to T.H.J.H.).     

Mechanism of Testosterone Deficiency in the Transgenic Sickle Cell Mouse

Testosterone deficiency is associated with sickle cell disease (SCD), but its underlying mechanism is not known. We investigated the possible occurrence and mechanism of testosterone deficiency in a mouse model of human SCD. Transgenic sickle male mice (Sickle) exhibited decreased serum and intratesticular testosterone and increased luteinizing hormone (LH) levels compared with wild type (WT) mice, indicating primary hypogonadism in Sickle mice. LH-, dbcAMP-, and pregnenolone- (but not 22-hydroxycholesterol)- stimulated testosterone production by Leydig cells isolated from the Sickle mouse testis was decreased compared to that of WT mice, implying defective Leydig cell steroidogenesis. There also was reduced protein expression of steroidogenic acute regulatory protein (STAR), but not cholesterol side-chain cleavage enzyme (P450scc), in the Sickle mouse testis. These data suggest that the capacity of P450scc to support testosterone production may be limited by the supply of cholesterol to the mitochondria in Sickle mice. The sickle mouse testis exhibited upregulated NADPH oxidase subunit gp91phox and increased oxidative stress, measured as 4-hydroxy-2-nonenal, and unchanged protein expression of an antioxidant glutathione peroxidase-1. Mice heterozygous for the human sickle globin (Hemi) exhibited intermediate hypogonadal changes between those of WT and Sickle mice. These results demonstrate that testosterone deficiency occurs in Sickle mice, mimicking the human condition. The defects in the Leydig cell steroidogenic pathway in Sickle mice, mainly due to reduced availability of cholesterol for testosterone production, may be related to NADPH oxidase-derived oxidative stress. Our findings suggest that targeting testicular oxidative stress or steroidogenesis mechanisms in SCD offers a potential treatment for improving phenotypic changes associated with testosterone deficiency in this disease.     

The effects of cold acclimation and nitric oxide on antioxidative enzymes in rat pancreas

Alterations of pancreatic antioxidative defense (AD) and possible nitric oxide (NO) role in AD organization of adult rats receiving l-arginine.HCl (2.25%) or N(omega)-nitro-l-arginine methyl ester (L-NAME.HCl, 0.01%) as drinking liquids and maintained at room (22+/-1 degrees C) or low (4+/-1 degrees C) temperature for 45 days were studied. For that purpose, copper, zinc- and manganese superoxide dismutase (CuZnSOD, MnSOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and glutathione reductase (GR) activities were determined. Cold-induced decrease of CuZnSOD was inhibited with L-NAME, while l-arginine produced the same effect as cold in both supplemented groups. Cold acclimation elevated GSH-Px activity. l-Arginine and L-NAME expressed no effect on GSH-Px in rats kept at room temperature. L-NAME additionally elevated cold-induced GSH-Px activity, l-arginine expressing a similar trend. Cold-induced increase in GST activity was inhibited by L-NAME, while l-arginine inhibited this enzyme in both supplemented groups. Cold acclimation increased GR activity in control and L-NAME-treated group and l-arginine expressed a similar trend. Neither of the treatments affected MnSOD and CAT activities. Cold-induced changes of pancreatic AD were additionally affected by the alterations in l-arginine-NO-producing pathway. Some AD changes in the same direction with l-arginine or L-NAME point to the complexity of nitrogen compounds metabolism and function, accompanied by tissue-specific response.