Kinesin‐14 is Important for Chromosome Segregation During Mitosis and Meiosis in the Ciliate Tetrahymena thermophila

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin‐14 is a conserved minus‐end directed microtubule motor that cross‐links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin‐14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP‐tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin‐14 is important for nuclear divisions that involve a bipolar spindle.     

Xanthoferrin,the α‐hydroxycarboxylate‐type siderophore of Xanthomonas campestris pv. campestris,is required for optimum virulence and growth inside cabbage

Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss (Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin‐type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α‐hydroxycarboxylate‐type siderophore (named xanthoferrin), which is required for growth under low‐iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low‐iron conditions. In contrast, the siderophore utilization ΔxsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore–iron complex. Our radiolabelled iron uptake studies confirm that the ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe³⁺) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin–Fe³⁺ complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low‐iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of ΔxssA and ΔxsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe³⁺.     

Identification of Pak1 inhibitors using water thermodynamic analysis

Abstract

p21-activated kinases (Paks) play an integral component in various cellular diverse processes. The full activation of Pak is dependent upon several serine residues present in the N-terminal region, a threonine present at the activation loop, and finally the phosphorylation of these residues ensure the complete activation of Pak1. The present study deals with the identification of novel potent candidates of Pak1 using computational methods as anti-cancer compounds. A diverse energy based pharmacophore (e-pharmacophore) was developed using four co-crystal inhibitors of Pak1 having pharmacophore features of 5 (DRDRR), 6 (DRHADR), and 7 (RRARDRP and DRRDADH) hypotheses. These models were used for rigorous screening against e-molecule database. The obtained hits were filtered using ADME/T and molecular docking to identify the high affinity binders. These hits were subjected to hierarchical clustering using dendritic fingerprint inorder to identify structurally diverse molecules. The diverse hits were scored against generated water maps to obtain WM/MM ΔG binding energy. Furthermore, molecular dynamics simulation and density functional theory calculations were performed on the final hits to understand the stability of the complexes. Five structurally diverse novel Pak1 inhibitors (4835785, 32198676, 32407813, 76038049, and 32945545) were obtained from virtual screening, water thermodynamics and WM/MM ΔG binding energy. All hits revealed similar mode of binding pattern with the hinge region residues replacing the unstable water molecules in the binding site. The obtained novel hits could be used as a platform to design potent drugs that could be experimentally tested against cancer patients having increased Pak1 expression.     

A study on neuroinflammatory marker in brain areas of okadaic acid (ICV) induced memory impaired rats

AimsThe aim of the present study is to investigate the status of proinflammatory cytokine in the brain of intracerebroventricular (ICV) okadaic acid (OKA) induced memory impaired rat.Main methodsOKA (200 ng) intracerebroventricular (ICV) was administered in rats. Memory was assessed by Morris water maze test. Biochemical marker of neuroinflammation (TNF-α, IL-β), total nitrite, mRNA (RT PCR) and protein expression (WB) of iNOS and nNOS were estimated in rat brain areas.Key findingsOKA caused memory-impairment in rats with increased expression of proinflammatory cytokine TNF-α and IL-1β and total nitrite in brain regions hippocampus and cortex. The expression of mRNA and protein of iNOS was increased while; the expressions were decreased in case of nNOS. Pretreatment with antidementic drugs donepezil (5 mg/kg, p.o.) and memantine (10 mg/kg, p.o) for 13 days protected ICV OKA induced memory impairment and changes in level of TNF-α, IL-β, total nitrite and expressions of iNOS and nNOS in OKA treated rat.SignificanceThis study suggests that neuroinflammation may play a vital role in OKA induced memory impairment.     

Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver

Smads are intracellular signaling mediators. Complexes of Smad2 and Smad3 with Smad4 transmit transforming growth factor-beta (TGF-β) receptor-induced signaling. Snail plays important roles in mesoderm formation, gastrulation, neural crest development, and epithelial mesenchymal transition. However, it remains unknown whether Smad3 and Snail expression is circadian rhythm-dependent. Here, we showed for the first time that Smad3 and Snail show circadian expression in human gingival fibroblasts (HGF-1) and human mesenchymal stem cells (MSC) after serum shock. They also showed circadian expression in the mouse liver. We confirmed that BMAL1/2, DEC1/2, VEGF, and PER1/2/3 also show circadian expression in both HGF-1 and MSC. The mRNA peaks and phases in circadian expression of these genes differed between HGF-1 and MSC. In a luciferase assay, Smad3 promoter activity was upregulated by CLOCK/BMAL1. These findings suggest that Smad3 and Snail have circadian rhythm in vitro and vivo, and that circadian expression of Smad3 depends on CLOCK/BMAL1.     

Oxygen Consumption and Respiration Following Two Yoga Relaxation Techniques

The present study was conducted to evaluate a statement in ancient yoga texts that suggests that a combination of both calming and stimulating measures may be especially helpful in reaching a state of mental equilibrium. Two yoga practices, one combining calming and stimulating measures (cyclic meditation) and the other, a calming technique (shavasan), were compared. The oxygen consumption, breath rate, and breath volume of 40 male volunteers (group mean ± SD, 27.0 ± 5.7 years) were assessed before and after sessions of cyclic meditation (CM) and before and after sessions of shavasan (SH). The 2 sessions (CM, SH) were 1 day apart. Cyclic meditation includes the practice of yoga postures interspersed with periods of supine relaxation. During SH the subject lies in a supine position throughout the practice. There was a significant decrease in the amount of oxygen consumed and in breath rate and an increase in breath volume after both types of sessions (2-factor ANOVA, paired t test). However, the magnitude of change on all 3 measures was greater after CM: (1) Oxygen consumption decreased 32.1% after CM compared with 10.1% after SH; (2) breath rate decreased 18.0% after CM and 15.2% after SH; and (3) breath volume increased 28.8% after CM and 15.9% after SH. These results support the idea that a combination of yoga postures interspersed with relaxation reduces arousal more than relaxation alone does.     

5-Membered NH...N hydrogen bonded molecular scaffold in a model dipeptide containing 3-aminophenylacetic acid: Crystal and solution conformations

Crystallographic and spectroscopic studies of a model dipeptidecontaining unusual amino acid residues establish the presence ofan intramolecular, 5-membered NH...N hydrogen bond involvingan amide NH (from 3-amino phenyl acetic acid residue) and anamide N atom from an adjacent amino acid residue in solid stateand in solution. The dipeptide also forms an infinite -sheet ribbon structure in crystals.     

Hypocholesterolemic effect of anhydrous milk fat ghee is mediated by increasing the secretion of biliary lipids

The anhydrous milk fat ghee is one of the important sources of fat in the Indian diet. Our earlier studies showed that rats fed diets containing greater than 2.5 wt% of ghee had lower levels of serum cholesterol compared with rats fed diets containing groundnut oil. To evaluate the mechanism of the hypocholesterolemic effect of ghee, male Wistar rats were fed a diet containing 2.5 or 5.0 wt% ghee for a period of 8 weeks. The diets were made isocaloric with groundnut oil. Both native and ghee heated at 120 degrees C containing oxidized lipids were included in the diet. The ghee in the diet did not affect the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity in the liver microsomes, but it significantly increased biliary excretion of cholesterol, bile acids, uronic acid, and phospholipids. The rats fed ghee had lower levels of cholesterol esters in the serum as well as in the intestinal mucosa. Both native and oxidized ghee influenced cholesterol metabolism. These results indicate that supplementation of diets with ghee lipids would increase the excretion of bile constituents and lower serum cholesterol levels.