CCL5 modulates pneumococcal immunity and carriage

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Recently, it has been shown that genetic polymorphisms can result in diminished expression of CCL5, which results in increased susceptibility to and progression of infectious diseases. We show that CCL5, together with Th cytokine mRNA expression, is temporally up-regulated during pneumococcal carriage. To determine the contribution of CCL5 to pneumococcal surface antigen A-specific humoral and cellular pneumococcal immunity, mice were treated with anti-CCL5 or control Abs before and during Streptococcus pneumoniae strain EF3030-challenge for the initiation of carriage. CCL5 blockade resulted in a decrease of CD4(+) and CD8(+) T cells as well as CD11b(+) cells in the spleen, cervical lymph node, lung, and nasopharyngeal associated lymphoid tissue during the recognition phase of the pneumococcal adaptive immune response. CCL5 blockade significantly reduced the Ag-specific IgG2a and IgG1 Abs in serum and IgA Ab levels in nasal washes. These decreases also corresponded to reductions in Ag-specific T cell (mucosal and systemic) responses. CCL5 inhibition resulted in decreasing the quantity of IL-4- and IFN-gamma-secreting CD4(+) T cells and increasing the number of Ag-specific IL-10-producing CD4(+) T cells; these changes combined also corresponded with the transition from pneumococcal carriage to lethal pneumonia. These data suggest that CCL5 is an essential factor for the induction and maintenance of protective pneumococcal immunity.     

Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1

PKCdelta has been shown to be activated by insulin and to interact with insulin receptor and IRS. PKB(Akt) plays an important role in glucose transport and glycogen synthesis. In this study, we investigated the possibility that PKCdelta may be involved in insulin-induced activation of PKB. Studies were conducted on primary cultures of rat skeletal muscle. PKB was activated by insulin stimulation within 5min and reached a peak by 15-30min. Insulin also increased the physical association between PKCdelta with PKB and with PDK1. The insulin-induced PKCdelta-PKB association was PI3K dependent. PKB-PKCdelta association was accounted for by the involvement of PDK1. Overexpression of dominant negative PKCdelta abrogated insulin-induced association of PKCdelta with both PKB and PDK1. Blockade of PKCdelta also decreased insulin-induced Thr308 PKB phosphorylation and PKB translocation. Moreover, PKCdelta inhibition reduced insulin-induced GSK3 phosphorylation. The results indicate that insulin-activated PKCdelta interacts with PDK1 to regulate PKB.     

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS.     

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Lack of association of colonic epithelium telomere length and oxidative DNA damage in Type 2 diabetes under good metabolic control

Background

Telomeres are DNA repeat sequences necessary for DNA replication which shorten at cell division at a rate directly related to levels of oxidative stress. Critical telomere shortening predisposes to cell senescence and to epithelial malignancies. Type 2 diabetes is characterised by increased oxidative DNA damage, telomere attrition, and an increased risk of colonic malignancy. We hypothesised that the colonic mucosa in Type 2 diabetes would be characterised by increased DNA damage and telomere shortening.

Methods

We examined telomere length (by flow fluorescent in situ hybridization) and oxidative DNA damage (flow cytometry of 8 – oxoguanosine) in the colonic mucosal cells of subjects with type 2 diabetes (n = 10; mean age 62.2 years, mean HbA1c 6.9%) and 22 matched control subjects. No colonic pathology was apparent in these subjects at routine gastrointestinal investigations.

Results

Mean colonic epithelial telomere length in the diabetes group was not significantly different from controls (10.6 [3.6] vs. 12.1 [3.4] Molecular Equivalent of Soluble Fluorochrome Units [MESF]; P = 0.5). Levels of oxidative DNA damage were similar in both T2DM and control groups (2.6 [0.6] vs. 2.5 [0.6] Mean Fluorescent Intensity [MFI]; P = 0.7). There was no significant relationship between oxidative DNA damage and telomere length in either group (both p > 0.1).

Conclusion

Colonic epithelium in Type 2 diabetes does not differ significantly from control colonic epithelium in oxidative DNA damage or telomere length. There is no evidence in this study for increased oxidative DNA damage or significant telomere attrition in colonic mucosa as a carcinogenic mechanism.     

Early Signals in Serum-Induced Increases in Ouabain-Sensitive Na+-K+ Pump Activity and in Glucose Transport in Rat Skeletal Muscle Are Amiloride-Sensitive

Abstract: The acute effects of serum on sodium-potassium (Na⁺-K⁺) pump activity and glucose uptake in cultured rat skeletal muscle were studied. Addition of serum to myo-tubes in phosphate-buffered saline caused Na⁺-K⁺ pump activity (as measured by changes in the ouabain-sensitive component of both membrane potential and ⁸⁶Rb uptake) to increase, with peak effects obtained after 30 min. The effect was blocked completely by treatment with amiloride, but not by tetrodotoxin, which blocks voltage-dependent Na⁺ channels. On transfer of myotubes to Na⁺-free, choline buffer, resting Na⁺-K⁺ pump activity decreased to about 10% of that in phosphate-buffered saline. Addition of regular serum, but not Na⁺-free serum, caused Na⁺-K⁺ pump activity to increase slightly. Similar results were obtained with serum on glucose uptake, the peak effect being reached within 15 min. Stimulation of glucose uptake by serum was partially reduced by amiloride and was not altered by tetrodotoxin. Removal of external Na⁺ also eliminated serum effects on glucose uptake. The results demonstrate that there are similar signals involving Na⁺-H⁺ exchange for serum-induced increases in Na⁺-K⁺ pump activity and glucose transport. The lack of complete blockade of serum-induced elevation of glucose transport suggests an additional, as yet undefined, intracellular signal for stimulation of this transport system.     

Variation and similarities in pollen features in some basal angiosperms, with some taxonomic implications

Species within three families of basal angiosperms (Trimeniaceae, Winteraceae, Monimiaceae) illustrate differences and similarities in pollen within a species, between species and between genera. Trimenia papuana (Trimeniaceae) has dimorphic pollen (inaperturate, polyforate), each confined to different individual plants. Other species have either disulculate or polyforate pollen. Evolution seems to be from disulculate to inaperturate to polyforate. Present-day Winteraceae have pollen in permanent tetrads except four species of Zygogynum with monads. Why? Did such monads appear as fossils before tetrads in Winteraceae? Molecular studies of Takhtajania perrieri indicate it is basal but its unique bicarpellate unilocular gynoecium seems derived. Although Hedycarya arborea and Kibaropsis caledonica have near-identical permanent pollen tetrads, many other features are very different. Hedycarya species have permanent tetrads or inaperturate monads with spinulose, `starry' or other sculpturing, and it is suggested this and recent molecular data indicate further studies are needed to determine generic limits.