Species identification and phylogenetic analysis of Leishmania isolated from patients,vectors and hares in the Xinjiang Autonomous Region,The People’s Republic of China

BackgroundVisceral leishmaniasis (VL) has been declared as one of the six major tropical diseases by the World Health Organization. This disease has been successfully controlled in China, except for some areas in the western region, such as the Xinjiang Autonomous Region, where both anthroponotic VL (AVL) and desert type zoonotic VL (DT-ZVL) remain endemic with sporadic epidemics.Methodology/Principal findingsHere, an eleven-year survey (2004–2014) of Leishmania species, encompassing both VL types isolated from patients, sand-fly vectors and Tarim hares (Lepus yarkandensis) from the Xinjiang Autonomous Region was conducted, with a special emphasis on the hares as a potential reservoir animal for DT-ZVL. Key diagnostic genes, ITS1, hsp70 and nagt (encoding N-acetylglucosamine-1-phosphate transferase) were used for phylogenetic analyses, placing all Xinjiang isolates into one clade of the L. donovani complex. Unexpectedly, AVL isolates were found to be closely related to L. infantum, while DT-ZVL isolates were closer to L. donovani. Unrooted parsimony networks of haplotypes for these isolates also revealed their relationship.Conclusions/SignificanceThe above analyses of the DT-ZVL isolates suggested their geographic isolation and independent evolution. The sequence identity of isolates from patients, vectors and the Tarim hares in a single DT-ZVL site provides strong evidence in support of this species as an animal reservoir.     

Microtubule assembly after treatment of pig oocytes with taxol: correlation with chromosomes, gamma-tubulin, and MAP kinase.

In this study, taxol was used as a tool to study the correlation of microtubule assembly with chromosomes, gamma-tubulin and phosphorylated mitogen-activated protein (MAP) kinase in pig oocytes at different maturational stages. Taxol treatment did not affect meiotic resumption and chromosome condensation but inhibited/disrupted chromosome alignment at the metaphase plate and bipolar spindle formation and thus meiotic progression. Microtubules were co-localized with chromosomes and were found to emanate from the chromosomes in taxol-treated oocytes, suggesting that chromosomes may serve as a source of microtubule organization. In addition, the concentric emanation of microtubules within the chromosome-surrounded area in taxol-treated oocytes suggests that microtubule emanation from the chromosomes may be directed by other microtubule-organizing material. The formation of one large spindle or >/=2 spindles in oocytes after taxol removal shows that minus end microtubule-organizing material can be normally located on both sides of chromosomes only when the chromosomes are aligned on the metaphase plate. The co-localization of gamma-tubulin and phosphorylated MAP kinase with microtubule assembly in both control and taxol-treated oocytes suggests that these two proteins are associated microtubule-nucleating material in pig oocytes. However, Western blot analysis showed that neither cytoplasmic microtubule aster formation nor extensive microtubule assembly in the chromosome region induced by taxol was caused by super-activation of MAP kinase. Taxol also induced microtubule assembly depending on chromosome distribution in the first polar body. The results suggest that chromosomes are always co-localized with microtubules and that emanation of microtubules from the chromosomes may be regulated/directed by microtubule-organizing material including gamma-tubulin and phosphorylated MAP kinase in pig oocytes.     

Molecular characterization of a patient with del(1)(q23–q25)

Summary We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23–1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23–q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000–16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21–1q25.     

Cystathionine β-Synthase Inhibition Is a Potential Therapeutic Approach to Treatment of Ischemic Injury

Hydrogen sulfide (H₂S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine β-synthase (CBS) has been implicated as the predominant H₂S-producing enzyme in central nervous system. When SH-SY5Y cells were transfected to overexpress CBS, these cells were able to synthesize H₂S when exposed to high levels of enzyme substrates but not substrate concentrations that may reflect normal physiological conditions. At the same time, these cells demonstrated exacerbated cell death when subjected to oxygen and glucose deprivation (OGD) together with high substrate concentrations, indicating that H₂S production has a detrimental effect on cell survival. This effect could be abolished by CBS inhibition. The same effect was observed with primary astrocytes exposed to OGD and high substrates or sodium hydrosulfide. In addition, CBS was upregulated and activated by truncation in primary astrocytes subjected to OGD. When rats were subjected to permanent middle cerebral artery occlusion, CBS activation was also observed. These results imply that in acute ischemic conditions, CBS is upregulated and activated by truncation causing an increased production of H₂S, which exacerbate the ischemic injuries. Therefore, CBS inhibition may be a viable approach to stroke treatment.     

Systemic hemodynamic function in humans with type 1 diabetes treated with protein kinase Cβ inhibition and renin-angiotensin system blockade: a pilot study

The protein kinase Cβ (PKCβ) system has been implicated in the deleterious vascular responses to hyperglycemia and angiotensin II (Ang?II) in experimental models of diabetes (DM). Whether these interactions are important in humans is unknown. Flow-mediated vasodilatation (FMD) was measured during clamped euglycemia and hyperglycemia, before and after randomization to PKCβ inhibition (ruboxistaurin; RBX, 32?mg daily, n = 13) or a placebo (n = 7) for 8?weeks in renin-angiotensin system (RAS) blockade-treated subjects with type 1 DM. Blood pressure responses to infused Ang?II were measured before and after randomization to RBX or a placebo. The RBX and placebo groups displayed similar clinical characteristics. Before RBX, FMD declined in response to hyperglycemia (6.8%?± 2.8% to 4.9%?± 1.8%). This effect was reversed after treatment with RBX (5.6%?± 3.1% to 6.0%?± 1.6% (within-group change, p = 0.009 (ANOVA)). No changes were observed in the placebo group. Infused Ang?II was associated with hypertensive responses in the RBX and placebo groups (p?< 0.05 (ANOVA)), and RBX did not influence this effect. In conclusion, RBX blunted the effect of hyperglycemia on FMD, suggesting that PKCβ may modulate endothelial function in type 1 DM. The lack of effect on Ang?II responses suggests that PKCβ inhibition may act through non-RAS pathways in humans with DM.     

K-RAS Mutant Pancreatic Tumors Show Higher Sensitivity to MEK than to PI3K Inhibition In Vivo

Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.     

Distribution of bovine fetuin and albumin in plasma, allantoic and amniotic fluids during development

A radioimmunoassay for bovine fetuin was developed and its specificity and validity established. Albumin was measured by radial-immunodiffusion assay. Fetuin levels in fetal plasma increased from 10 to 15 mg/ml between 4 and 8 months of gestation; albumin levels remained higher than fetuin. Neonatal plasma fetuin levels rapidly declined during the first 14 days post partum, coincident with a marked reciprocal increase in albumin levels. In allantoic fluid fetuin and albumin concentrations reached a peak at 7 months but fetuin values were always higher. In amniotic fluid both proteins peaked at 8 months; albumin levels were similar to those in allantoic fluid but fetuin values remained consistently lower than those in allantoic fluid throughout gestation. Fetuin levels in maternal plasma declined from 0.7 to 0.4 mg/ml between 1 month and term. We conclude that (1) at term there is an abrupt change from fetuin synthesis to increased albumin synthesis by the neonatal liver; (2) fetuin appears to be preferentially accumulated in the allantois whereas albumin is equally concentrated in the allantois and amnion.     

Potent inhibition of human leukocyte elastase by 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamide derivatives

The design, synthesis, and in vitro biochemical evaluation of a class of mechanism-based inhibitors of human leukocyte elastase (HLE) that incorporate in their structure a 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold with appropriate recognition and reactivity elements appended to it is described. The synthesized compounds were found to be efficient, time-dependent inhibitors of HLE. The interaction of the inhibitors with HLE is postulated to lead to the formation of a highly reactive N-sulfonyl imine (a Michael acceptor) that arises from an enzyme-induced sulfonamide fragmentation cascade. Subsequent reaction ultimately leads to the formation of a relatively stable acyl enzyme. The results cited herein demonstrate convincingly the superiority of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold over other scaffolds (e.g., saccharin) in the design of inhibitors of (chymo)trypsin-like serine proteases.     

The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta

The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.