Binding of human centrin 2 to the centrosomal protein hSfi1

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat. The two standard peptides bind human centrin 2 and its isolated C-terminal domain with high affinity (approximately 10(7) M(-1)) by an enthalpy-driven mechanism, with a moderate Ca2+ dependence. The Pro-containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C-terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well-conserved Trp residue situated in the C-terminus of each repeat is deeply embedded in a protein hydrophobic cavity, indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this hypothesis and previous studies on centrin self-assembly, we propose a working model for the role of centrin-Sfi1 interactions in the dynamic structure of centrosome-associated contractile fibers.     

Work ability of health care shift workers: What matters?

This paper aims at identifying variables associated with inadequate work ability among nursing personnel at a public hospital, considering factors related to socio-demographic, lifestyles, working conditions, and health outcomes. A cross-sectional study was conducted in a university hospital in S?o Paulo, Brazil, as part of a larger research study on tolerance to 12 h night work. Nursing staff included registered nurses, nurse technicians, and nurse aides; in total, there were 996 healthcare workers (878 female; 118 male) at the time of the study. Some 696 workers (69.9%) of the population agreed to participate. Data collection (October 2004-July 2005) was based on a comprehensive questionnaire about living and working conditions (including incivility at work, work demands, work control, and support), mental and physical health symptoms (fatigue and sleep problems), and work ability. This report presents analyses of the adapted Brazilian version of the Work Ability Index (WAI) and associated variables. The study population worked one of the following shift schedules at this hospital: 12 h nights followed by 36 h off or 9 h or 6 h day (morning or afternoon) shifts. The mean age of the respondents was 34.9 (S.D.+/-10.4) years of age; 31.5% of the participants held two jobs. Statistical analyses using a hierarchical multiple logistic regression model were performed to evaluate the factors associated with inadequate (moderate and low scores) of the WAI. The significantly associated factors were socio-demographic (income responsibility, sole breadwinner, raising kids, age group), working conditions (thermal discomfort, organization of the workplace, and verbal abuse), and health outcomes (high body mass index, obesity, sleep problems, and fatigue). In spite of limitations of the study design, results indicate that the nursing profession is associated with stressful working conditions, contributing to inadequate WAI. This is in addition to bad living conditions and precarious work. Intervention measures, either at the workplace or at individual levels, are necessary to prevent a decrease in work ability, even in this quite young working population.     

Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.     

Sulfated HNK-1 epitope in developing and mature kidney: a new marker for thin ascending loop of Henle and tubular injury in acute tubular necrosis.

The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle.     

Effects of tumor necrosis factor antagonist treatment on hepatitis C-related immunological abnormalities

BACKGROUND: Chronic hepatitis C infection is frequently associated with a mixed cryoglobulinaemia and circulating auto-antibodies, especially anti-smooth muscle cells (SMA) and anti-liver/kidney/microsome type 1 (LKM-1) anti-tissue antibodies. Treatments with TNF antagonists favour the emergence of auto-antibodies, and particularly anti-dsDNA antibodies. OBJECTIVE: To determine the impact of TNF antagonists on hepatitis C-related immune abnormalities. METHODS: We prospectively monitored for 14 weeks, six patients with actively replicating chronic hepatitis C, initiating an anti-TNF treatment for an associated rheumatoid arthritis. RESULTS: Anti-nuclear and anti-dsDNA antibodies were induced in two and three patients, respectively. Treatment had no impact on the production of antibodies against extractable nuclear antigens, and it did not induce anti-tissues antibodies in any patient. Cryoglobulinaemia appeared in 2/6 patients, and it persisted in 2 others. No patient developed any news signs of autoimmunity. HCV viraemia remained unchanged. CONCLUSIONS: Induction of auto-antibodies by TNF antagonist treatments does not involve anti-tissues antibodies, even in patients with actively replicating chronic hepatitis C prone to produce anti-SMA and anti-LKM-1 antibodies. In contrast, TNF antagonists may favour emergence of cryoglobulinaemia in such patients.     

Uncovering conserved patterns in bioactive peptides in Metazoa

Bioactive (neuro)peptides play critical roles in regulating most biological processes in animals. Peptides belonging to the same family are characterized by a typical sequence pattern that is conserved among the family's peptide members. Such a conserved pattern or motif usually corresponds to the functionally important part of the biologically active peptide. In this paper, all known bioactive (neuro)peptides annotated in Swiss-Prot and TrEMBL protein databases are collected, and the pattern searching program Pratt is used to search these unaligned peptide sequences for conserved patterns. The obtained patterns are then refined by combining the information on amino acids at important functional sites collected from the literature. All the identified patterns are further tested by scanning them against Swiss-Prot and TrEMBL protein databases. The diagnostic power of each pattern is validated by the fact that any annotated protein from Swiss-Prot and TrEMBL that contains one of the established patterns, is indeed a known (neuro)peptide precursor. We discovered 155 novel peptide patterns in addition to the 56 established ones in the PROSITE database. All the patterns cover 110 peptide families. Fifty-five of these families are not characterized by the PROSITE signatures, and 12 are also not identified by other existing motif databases, such as Pfam and SMART. Using the newly identified peptide signatures as a search tool, we predicted 95 hypothetical proteins as putative peptide precursors.     

The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae

The very first member of K(+) channels toxins from the venom of the Iranian scorpion Odonthobuthus doriae (OdK1) was purified, sequenced and characterized physiologically. OdK1 has 29 amino acids, six conserved cysteines and a pI value of 4.95. Based on multiple sequence alignments, OdK1 was classified as alpha-KTx 8.5. The pharmacological effects of OdK1 were studied on six different cloned K(+) channels (vertebrate Kv1.1-Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183+/-3 nM but did not affect any of the other channels.     

Use of SANS and biophysical techniques to reveal subtle conformational differences between native apo-calmodulin and its unfolded states

Apo-calmodulin, a small, mainly α, soluble protein is a calcium-dependent protein activator. It is made of two N- and C-terminal domains having a sequence homology of 70%, an identical folding but different stabilities, and is thus an interesting system for unfolding studies. The use of small angle neutron scattering (SANS) and other biophysical techniques has permitted to reveal conformational difference between native and thermal denatured states of apo-calmodulin. The results show that secondary and tertiary structures of apo-calmodulin evolve in a synchronous way, indicating the absence in the unfolding pathway of molten-globule state sufficiently stable to affect transition curves. From SANS experiments, at 85 °C, apo-calmodulin adopts a polymer chain conformation with some residual local structures. After cooling down, apo-calmodulin recovers a compact state, with a secondary structure close to the native one but with a higher radius of gyration and a different tyrosine environment. In fact on a timescale of few minutes, heat denaturation of apo-calmodulin is partially reversible, but on a time scale of hours (for SANS experiments), the long exposure to heat may lead to a non-reversibility due to some chemical perturbation of the protein. In fact, from Mass Spectrometry measurements, we got evidence of dehydration and deamidation of heated apo-calmodulin.     

Molecular cloning and characterization of a novel RING zinc-finger protein gene up-regulated under in vitro salt stress in cassava

Cassava (Manihot esculenta Crantz) is one of the world’s most important food crops. It is cultivated mainly in developing countries of tropics, since its root is a major source of calories for low-income people due to its high productivity and resistance to many abiotic and biotic factors. A previous study has identified a partial cDNA sequence coding for a putative RING zinc finger in cassava storage root. The RING zinc finger protein is a specialized type of zinc finger protein found in many organisms. Here, we isolated the full-length cDNA sequence coding for M. esculenta RZF (MeRZF) protein by a combination of 5′ and 3′ RACE assays. BLAST analysis showed that its deduced amino acid sequence has a high level of similarity to plant proteins of RZF family. MeRZF protein contains a signature sequence motif for a RING zinc finger at its C-terminal region. In addition, this protein showed a histidine residue at the fifth coordination site, likely belonging to the RING-H2 subgroup, as confirmed by our phylogenetic analysis. There is also a transmembrane domain in its N-terminal region. Finally, semi-quantitative RT-PCR assays showed that MeRZF expression is increased in detached leaves treated with sodium chloride. Here, we report the first evidence of a RING zinc finger gene of cassava showing potential role in response to salt stress.     

AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase

Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.