csi2p modulates microtubule dynamics and organizes the bipolar spindle for chromosome segregation

Proper chromosome segregation is of paramount importance for proper genetic inheritance. Defects in chromosome segregation can lead to aneuploidy, which is a hallmark of cancer cells. Eukaryotic chromosome segregation is accomplished by the bipolar spindle. Additional mechanisms, such as the spindle assembly checkpoint and centromere positioning, further help to ensure complete segregation fidelity. Here we present the fission yeast csi2⁺. csi2p localizes to the spindle poles, where it regulates mitotic microtubule dynamics, bipolar spindle formation, and subsequent chromosome segregation. csi2 deletion (csi2Δ) results in abnormally long mitotic microtubules, high rate of transient monopolar spindles, and subsequent high rate of chromosome segregation defects. Because csi2Δ has multiple phenotypes, it enables estimates of the relative contribution of the different mechanisms to the overall chromosome segregation process. Centromere positioning, microtubule dynamics, and bipolar spindle formation can all contribute to chromosome segregation. However, the major determinant of chromosome segregation defects in fission yeast may be microtubule dynamic defects.     

Pyridoxal 5'-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B₆ requiring enzymes. PLP reacts with apo-B₆ enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B₆ enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B₆ proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B₆ enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.     

Age, photoperiod and estrogen dependent variations in the shell gland and the expression of AVT in the ovary of Japanese quail

Present work was undertaken to describe (i) age dependent (prepuberal-3, 4, 5 and 6 weeks old, puberal and actively laying 8 and 12 weeks old and aged 78 weeks old) (ii) photoperiodic response dependent (photosensitive and photorefractory) and sex steroid dependent (estradiol benzoate and its antagonist tamoxifen treated) variation in the ovary and shell gland activity of Japanese quail (Coturnix coturnix japonica). Further, in view of the role of neurohypophysial peptide arginine vasotocin (AVT) in many physiological processes including age/reproduction related oviposition, expression of ir-AVT was also monitored in the ovary of quail. All the parameters associated with histodifferentiation increased rapidly during the developing stages followed by a decrease in old age, which also increased in reproductively quiescent photorefractory birds following estradiol treatment and decreased in reproductively active photosensitive quail following tamoxifen treatment. Using AVT-specific antibody, expression of immunoreactive AVT (ir-AVT) observed in the ovary of photosensitive quail was not detected in the photorefractory quail. However, administration of estrogen in the photorefractory quail stimulated the growth and activity of ovary and shell gland also resulted in the expression of ovarian ir-AVT. On the other hand, tamoxifen eliminated the localization of ir-AVT in the ovary of photosensitive quail in addition to a decrease in the shell gland protein and alkaline phosphatase activity. It is concluded that estrogen not only affects the growth and differentiation of ovary and oviduct including shell gland but also regulates the expression of ovarian AVT. It is also suggested that in addition to reported paracrine effect of AVT in the shell gland of Japanese quail for oviposition, ovarian AVT may also affect ovarian function (ovulation), and in part, this regulation is estrogen dependent.     

Diabetes alters LDL receptor and PCSK9 expression in rat liver

Since the hepatic LDL receptor is regarded as a major determinant of plasma LDL levels, the effect of diabetes on the expression of this receptor was examined in rat liver. Inducing diabetes with streptozotocin caused a significant reduction in hepatic LDL receptor mRNA levels in concert with an increase in serum cholesterol levels. However, LDL receptor protein levels were unaffected by the diabetic state. Further investigation revealed that protein levels of PCSK9, which has been shown to enhance the degradation of the LDL receptor protein, were significantly decreased in the diabetic rats explaining the lack of reduction in LDL receptor protein levels. These observations indicate that the rate of LDL receptor cycling (function) in diabetic rats is decreased resulting in higher serum LDL levels.     

Temporal variations of the postnatal rat urinary proteome as a reflection of systemic maturation

The rat kidney matures during the first 2 wk of life, suggesting that temporal variations in the urinary proteome may occur during this period. We describe the urine proteome during postnatal development in the rat and demonstrate specific proteomic changes corresponding to developmental milestones. Urine was collected from 30 rats at five postnatal (P) days of life (P1, P3, P7, P14, and >P30) by bladder aspiration. The proteome was assessed by nano-ESI-LC-MS/MS. For identification, we used stringent criteria to provide a 1% false positive rate at the peptide level. The proteins in common at each time interval decreased during postnatal maturation. When comparing all five developmental times, six proteins were ubiquitously present. We detected 14 proteins involved with cellular adhesion, structure, or proliferation and differentiation only during neonatal development. Additionally, 30 proteins were specific to adults, of which 13 originated from the prostate or seminal vesicle. This is the first MS characterization of the normal urinary proteome in early postnatal rodent development that demonstrates distinct differences correlating with different stages of tissue maturation. Further characterization of the normal urinary proteome may provide the basis for identification of urinary biomarkers of diseases of the urinary tract.     

The plmS2-encoded cytochrome P450 monooxygenase mediates hydroxylation of phoslactomycin B in Streptomyces sp. strain HK803

Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS(2) open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS(2) ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS(2) ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS(2) converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS(2) was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS(2) (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS(2) (K(m) of 45.3 +/- 9.0 microM and k(cat) of 0.27 +/- 0.04 s(-1)) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.     

Higher accumulation of proteinase inhibitors in flowers than leaves and fruits as a possible basis for differential feeding preference of Helicoverpa armigera on tomato (Lycopersicon esculentum Mill, Cv. Dhanashree)

Tomato (Lycopersicon esculentum, Mill; cultivar- Dhanashree) proteinase inhibitors (PIs) were tested for their trypsin inhibitory (TI) and Helicoverpa armigera gut proteinases inhibitory (HGPI) activity in different organs of the tomato plants. Analysis of TI and HGPI distribution in various parts of the plant showed that flowers accumulated about 300 and 1000 times higher levels of TI while 700 and 400 times higher levels of HGPI as compared to those in leaves and fruits, respectively. Field observation that H. armigera larvae infest leaves and fruits but not the flowers could be at least partially attributed to the protective role-played by the higher levels of PIs in the flower tissue. Tomato PIs inhibited about 50-80% HGP activity of H. armigera larvae feeding on various host plants including tomato, of larvae exposed to non-host plant PIs and of various larval instars. Tomato PIs were found to be highly stable to insect proteinases wherein incubation of inhibitor with HGP even for 3h at optimum conditions did not affect inhibitory activity. Bioassay using H. armigera larvae fed on artificial diet containing tomato PIs revealed adverse effect on larval growth, pupae development, adult formation and fecundity.