Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

P2X7 receptor-Pannexin1 complex: pharmacology and signaling

Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X(7) receptor (P2X(7)R) activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X(7)R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X(7)R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X(7)R stimulation or by membrane depolarization was blocked by Panx1 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X(7)R antagonist, prevented current activation by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X(7)) peptides, provided evidence that the Src homology 3 death domain of the COOH-terminus of the P2X(7)R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 muM TAT-P2X(7) peptide caused 50% reduction in Src binding to the P2X(7)R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X(7) peptide, and by the Src tyrosine inhibitor PP2 and these compounds prevented both large-conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X(7)R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X(7)R.     

Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

Effects of an evaporative cooling system on plasma cortisol, IGF-I, and milk production in dairy cows in a tropical environment

Access to an evaporative cooling system can increase production in dairy cows because of improved thermal comfort. This study aimed to evaluate the impact of ambient temperature on thermoregulation, plasma cortisol, insulin-like growth factor 1 (IGF-I), and productive status, and to determine the efficiency of an evaporative cooling system on physiological responses under different weather patterns. A total of 28 Holstein cows were divided into two groups, one with and the other without access to a cooling system with fans and mist in the free stall. The parameters were analyzed during morning (0700 hours) and afternoon milking (1430 hours) under five different weather patterns throughout the year (fall, winter, spring, dry summer, and rainy summer). Rectal temperature (RT), body surface temperature (BS), base of tail temperature (TT), and respiratory frequency (RF) were lower in the morning (P?<?0.01). The cooling system did not affect RT, and both the groups had values below 38.56 over the year (P?=?0.11). Cortisol and IGF-I may have been influenced by the seasons, in opposite ways. Cortisol concentrations were higher in winter (P?<?0.05) and IGF-I was higher during spring-summer (P?<?0.05). The air temperature and the temperature humidity index showed positive moderate correlations to RT, BS, TT, and RF (P?<?0.001). The ambient temperature was found to have a positive correlation with the physiological variables, independent of the cooling system, but cooled animals exhibited higher milk production during spring and summer (P?<?0.01).     

Tasa estimada de disposición de glucosa en pacientes menores de 18 años con diabetes mellitus tipo 1 y sobrepeso-obesidad

ObjectiveTo assess the estimated glucose disposal rate (eGDR), insulin dose, and lipoprotein profile in children with type 1 diabetes mellitus (T1DM) and overweight or obesity as compared to children with T1DM and normal weight.MethodsA total of 115 patients (aged 5-16 years) with T1DM on intensive insulin therapy were recruited. The following parameters were measured: weight, height, body mass index, waist and hip circumference, insulin dose, eGDR, glycosylated hemoglobin, blood pressure, and lipoprotein profile. Results were stratified by sex and age.ResultsNo significant differences were found in eGDR between children with normal weight, overweight, and obesity. However, obese children older than 11 years had lower eGDR values (9.3 ± 1.3 vs 10.1 ± 0.8 mg kg^-1min^-1; p < 0.01). Insulin dose was higher in overweight and obese children, especially in IU/m²/day (37.7 vs 36.1 vs 29.4 respectively; p < 0.01). Obese children had higher low-density lipoprotein cholesterol levels than children with overweight and normal weight (106.5 vs 91.7 vs 91.5 mg/dL respectively; p < 0.01). No correlation was found between waist circumference and the different markers of insulin resistance.ConclusionsValues of eGDR values were lower in obese children with T1DM older than 11 years, and this may therefore be considered a marker of insulin resistance. Insulin dose was higher in diabetic patients with overweight or obesity, specially in IU/m²/day. Obese children with T1DM had a lipoprotein profile of cardiovascular risk.     

Atomic Resolution Cryo-EM Structure of β-Galactosidase

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Pilot clinical trial of type 1 dendritic cells loaded with autologous tumor lysates combined with GM-CSF, pegylated IFN, and cyclophosphamide for metastatic cancer patients

Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-α, TNF-α, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day -7), GM-CSF (days 1-4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-γ-ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [(111)In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.     

Tropical freshwater teleosts from Miocene beds of eastern Patagonia, southern Argentina

Loricariid and pimelodid-like siluriforms—along with undetermined percomorph—are described for the first time from Patagonia. Vertebrate fossils, including fish, mammals and birds, were found in beds attributed to the top of the Puerto Madryn Formation. These levels supposedly corresponded to the “Rionegrense marino” of former authors. Mammals occurring in the site suggest a Huayquerian age for the fossiliferous beds. The base of the Huayquerian was dated at about 9 Ma and the top is younger than 6.5 Ma. Radioisotopic dating in the marine shell beds of the Puerto Madryn Formation ranges from 11 to 9 Ma. Consequently, the section described here appears to be younger than the typical Puerto Madryn Formation from which it is separated by an unconformity. The section is correlated with the type Río Negro Formation from northern Patagonia, which also includes Huayquerian fossils. Freshwater fishes were previously unknown in beds younger than the middle Miocene in southern South America. This is also the southernmost record of loricariid fishes. The association of aquatic continental and terrestrial vertebrates indicates for the first time in Península Valdés beds of freshwater origin. The evidence apported by fossils is also in agreement with global climate trends. The local extinction of loricariids in Patagonia possibly occurred much later than the time of deposition.