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.     

Functional characterization of the copper-transporting P-type ATPase gene of <Emphasis Type="Italic">Penicillium janthinellum</Emphasis> strain GXCR

Copper (Cu)-transporting P-type ATPase (CTPA) genes have been documented to play an important role in resistance to heavy metals. However, our understanding of roles of CTPA genes of the filamentous fungi was based only on sequence similarity prediction before. In a previous study, we isolated a Penicillum janthinellum strain GXCR of higher tolerance to Cu (200 mM). In this study, we cloned the partial cDNA of CTPA gene, named PcpA, from the strain GXCR. Sequence alignment indicated that the cloned cDNA sequence has the highest identity (94.4%) with a predictive CTPA gene of Aspergillus clavatus. The PcpA-encoded protein, termed PcpA, has classical functional domains of CTPAs, and shows differences from reported CTPAs in some specific sequence motifs and transmembrane regions. Expression of the PcpA was induced by extracellular Cu, cadmium (Cd), and silver (Ag). PcpA RNA interference (RNAi) mutants with a reduced level of PcpA mRNA were more sensitive to Cu, iron, Cd, and Ag than the wild-type (WT) strain GXCR. When grown in the presence of Cu, iron, and Cd, intracellular Cu and iron contents in the PcpA RNAi mutant were significantly (P<0.05) lower than those in the WT; However, intracellular Cd content in the mutant was significantly (P<0.05) higher than that in the WT. Taken together, it can be concluded that the PcpA functions in Cu uptake and homeostasis, iron uptake, and Cd export from the cytosol to the extracytosol.     

The <Emphasis Type="Italic">Endo-β-Mannanase</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis</Emphasis>, rice,and poplar

Mannans are widespread hemicellulosic polysaccharides in plant cell walls. Hydrolysis of the internal β-1,4-d-mannopyranosyl linkage in the backbone of mannans is catalyzed by endo-β-mannanase. Plant endo-β-mannanase has been well studied for its function in seed germination. Its involvement in other plant biological processes, however, remains poorly characterized or elusive. The completed genome sequences of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and poplar (Populus trichocarpa) provide an opportunity to conduct comparative genomic analysis of endo-β-mannanase genes in these three species. In silico sequence analysis led to the identification of eight, nine and 11 endo-β-mannanase genes in the genomes of Arabidopsis, rice, and poplar, respectively. Sequence comparisons revealed the conserved amino acids and motifs that are critical for the active site of endo-β-mannanases. Intron/exon structure analysis in conjunction with phylogenetic analysis implied that both intron gain and intron loss has played roles in the evolution of endo-β-mannanase genes. The phylogenetic analysis that included the endo-β-mannanases from plants and other organisms implied that plant endo-β-mannanases have an ancient evolutionary origin. Comprehensive expression analysis of all Arabidopsis and rice endo-β-mannanase genes showed divergent expression patterns of individual genes, suggesting that the enzymes encoded by these genes, while carrying out the same biochemical reaction, are involved in diverse biological processes.     

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.