Glucose,cholesterol,total proteins and insulin-like growth factor typeI values in the follicular fluid of female river buffaloes (Bubalus bubalis)

Twenty six Murrah female river buffaloes, between 45 and 70 d post-partum, empty, multiparae, with an average live weight of 675 ± 56 kg, and average body condition of 3.5 points, in a 1 to 5 scale, were used to determine the concentrations of glucose, cholesterol, total protein and insulin-like growth factor type I(IGF-I) in the follicular fluid. The fluid was collected from dominant follicles, with diameters between 8 and 12 mm, by in vivo follicular aspiration. The oestrous cycle stage was not taken into account. The wave of follicular development was synchronized six days prior to the collection. Biochemical analyses of glucose and cholesterol were performed by the enzymatic colorimetric method with the utilization of commercial kits of Glicose (GOD-PAP) and Cholesterol (CHOD-PAP) (Kovalent), respectively. For the determination of total protein, the commercial kit total Protein (Kovalent), method Biuret, was employed. Readings were carried out through absorption spectrophotometry with visible light. Through the radioimmunoanalysis (RIA) technique the concentration of IGF-I was obtained using commercial kits of IRMA IGF-I (IMMUNOTECH). Descriptive statistics was used, by applying the PROC MEANS procedure of the SAS (2009) statistical package. Glucose concentrations (4.0 ± 0.75 mmol/L) and IGF-I (340 ± 129.83 ng/mL) showed higher values in female river buffaloes and dairy cows regarding those reported in other studies. However, cholesterol levels (0.51 ± 0.12 mmol/L) and total proteins (58.4 ± 4.43 g/L) were lower. Results indicate that there is a relationship between the concentration of biochemical indicators, the nutritional aspects, the diameter of the aspired follicles and the productive period.

Uncoupling proteins (UCP) in unicellular eukaryotes: True UCPs or UCP1-like acting proteins?

Uncoupling proteins belong to the superfamily of mitochondrial anion carriers. They are apparently present throughout the Eukarya domain in which only some members have an established physiological function, i.e. UCP1 from brown adipose tissue is involved in non-shivering thermogenesis. However, the proteins responsible for the phenotype observed in unicellular organisms have not been characterized. In this report we analyzed functional evidence concerning unicellular UCPs and found that true UCPs are restricted to some taxonomical groups while proteins conferring a UCP1-like phenotype to fungi and most protists are the result of a promiscuous activity exerted by other mitochondrial anion carriers. We describe a possible evolutionary route followed by these proteins by which they acquire this promiscuous mechanism. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Conjugation of Toll Like Receptor 7 agonist through conjugation to immunogenic proteins. Synthesis, characterization, formulation and pre-clinical evaluation

The next generation of vaccine adjuvant are represented by a wide ranging set of molecules called Toll like agonists (TLR’s). Although many of these molecules are complex structures extracted from microorganisms, small molecule TLR agonists have also been identified. However, delivery systems have not been optimized to allow their effective delivery in conjunction with antigens. Here we describe a novel approach in which a small molecule TLR agonist has been conjugated directly to antigens to ensure effective co delivery. We describe the conjugation of a relevant protein, a recombinant protective antigen from S.pneumoniae (RrgB), which is linked to a TLR7 agonist. Following thorough characterization to ensure there was no aggregation, the conjugate was evaluated in a murine infection model. Results showed that the conjugate extended animals’ survival after lethal challenge with S.pneumoniae. Comparable results were obtained with a 10 fold lower dose than that of the native unconjugated antigen. Notably, the animals immunized with the same dose of unconjugated TLR7 agonist and antigen showed no adjuvant effect. The increased immunogenicity was likely a consequence of the co-localization of TLR7 agonist and antigen by chemical binding and is was more effective than simple co-administration. Likely, this approach can be adopted to reduce the dose of antigen required to induce protective immunity, and potentially increase the safety of a broad variety of vaccine candidates

CopY-like copper inducible repressors are putative 'winged helix' proteins

CopY of Enterococcus hirae is a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the cop operon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutans have similar affinities not only for their native promoters, but also for heterologous cop promoters. CopZ of L. lactis accelerated the release of CopY from the promoter, suggesting that CopZ of L. lactis acts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureus and related repressors. BlaI and MecI have known structures and belong to the family of 'winged helix' proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactis showed that it contained the same alpha-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the 'winged helix' type.

Insulin-like growth factors (IGFs), IGF binding proteins, and other endocrine factors in milk: role in the newborn

The role of colostrum and milk in the neonate has been chiefly recognized as a comprehensive nutrient foodstuff. In addition, the provision of colostrum-the first milk-for early immune capacity has been well documented for several species. Colostrum is additionally a rich and concentrated source of various factors that demonstrate biological activity in vitro. Three hypotheses have been proposed for the phenotypic function of these secreted bioactive components: (1) only mammary disposal, (2) mammary cell regulation, and (3) neonatal function [gastrointestinal tract (GIT) or systemic]. Traditionally, it was assumed that the development of the GIT is preprogrammed and not influenced by events occurring in the intestinal lumen. However, a large volume of research has demonstrated that colostrum (or milk-borne) bioactive components can basically contribute to the regulation of GIT growth and differentiation, while their role in postnatal development at physiological concentrations has remained elusive. Much of our current understanding is derived from cell culture and laboratory animals, but experimentation with agriculturally important species is taking place. This chapter provides an overview of work conducted primarily in neonatal calves and secondarily in other species on the effects on neonates of selected peptide endocrine factors (hormones, growth factors, in part cytokines) in colostrum. The primary focus will be on insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) and other bioactive peptides, but new interest and concern about steroids (especially estrogens) in milk are considered as well.

Subcellular distribution of the insulin-like growth factor (IGF) binding proteins (IGFBPs) 2 and 3 in articular chondrocytes

The insulin-like growth factor (IGF) is a major anabolic regulator in articular cartilage. The IGF-binding proteins (IGFBPs) are increased during osteoarthritis (OA), but the function of the later proteins remains unknown. In general, the IGFBPs are pluripotential effectors capable of IGF regulation and of acting on their own to control key cell functions, including survival and proliferation. The independent functions are often associated with their cell location, and therefore this study explores the distribution of IGFBP-2 and IGFBP-3 in articular chondrocytes. Immunohistochemistry was used to localize IGFBP-2 in normal human articular cartilage. Bovine chondrocytes were used for subcellular fractionation (hypotonic cell lysis) under nonreducing conditions and nuclear purification (centrifugation on sucrose cushions). Cell fraction markers and IGFBPs were assayed in the subcellular fractions by Western immunoblot. The IHC results showed association of IGFBP-2 with chondrocytes, but not with the nuclei. Subcellular fractionation of isolated chondrocytes yielded intact nuclei as assessed at the light microscopic level; the nuclear marker histone H1 was exclusively associated with this fraction. More than 90% of the cytoplasmic marker GAPDH and all the detectable IGFBP-2 were in the cytoplasmic fraction. Immunoreactive IGFBP-3 was found in the cytoplasmic and peri-nuclear/nuclear fractions. Chondrocytes contain intracellular IGFBP-2 and IGFBP-3 but only IGFBP-3 is associated with nuclei. This suggests the hypothesis that the actions of these IGFBPs in articular cartilage extend beyond the classic modulation of IGF receptor action.

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

Nuclear proteins interacting with an AP-1/CRE-like promoter sequence in the human TNF$\alpha$ gene

Monocyte developmental heterogeneity is reflected at the cellular level by differential activation competence, at the molecular level by differential regulation of gene expression. LPS activates monocytes to produce tumor necrosis factor-$\alpha$ (TNF). Events occurring at the molecular level necessary for TNF regulation have not been elucidated, but depend both on activation signals and the maturation state of the cell: Peripheral blood monocytes produce TNF upon LPS stimulation, but only within the first 72 hours of culture. Expression of c-fos is associated with monocytic differentiation and activation; the fos-associated protein, c-jun, is also expressed during monocyte activation. Increased cAMP levels are associated with down regulation of macrophage function, including LPS-induced TNF transcription. Due to these associations, we studied a region of the TNF promoter which resembles the binding sites for both AP-1(fos/jun) and CRE-binding protein (or ATF) in order to identify potential molecular markers defining activation competent populations of monocytic cells.^ Nuclear protein binding studies using extracts from THP-1 monocytic cells stimulated with LPS, which stimulates, or dexamethasone (Dex) or pentoxyfilline (PTX), which inhibit TNF production, respectively, suggest that a low mobility doublet complex may be involved in regulation through this promoter region. PTX or Dex increase binding of these complexes equivalently over untreated cells; approximately two hours after LPS induction, the upper complex is undetectable. The upper complex is composed of ATF2 (CRE-BP1); the lower is a heterodimer of jun/ATF2. LPS induces c-jun and thus may enhance formation of jun-ATF2 complexes. The simultaneous presence of both complexes may reduce the amount of TNF transcription through competitive binding, while a loss of the upper (ATF2) and/or gain of the lower (jun-ATF2) allow increased transcription. AP-1 elements generally transduce signals involving PKC; the CRE mediates a cAMP response, involving PKA. Thus, this element has the potential of receiving signals through divergent signalling pathways. Our findings also suggest that cAMP-induced inhibition of macrophage functions may occur via down regulation of activation-associated genes through competitive binding of particular cAMP-responsive nuclear protein complexes. ^

Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein.

U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.

A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to Gβ5 subunits

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the α subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein γ subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different Gβ subunits reveals specific interaction between RGS11 and Gβ5. The expression of mRNA for RGS11 and Gβ5 in human tissues overlaps. The Gβ5/RGS11 heterodimer acts as a GAP on Gαo, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for Gα proteins and form complexes with specific Gβ subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.

Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein

In the current model for bacterial cell division, FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other proteins such as FtsA. This putative protein complex ultimately generates the division septum. Herein we report that FtsZ and FtsA proteins tagged with green fluorescent protein (GFP) colocalize to division-site ring-like structures in living bacterial cells in a visible space between the segregated nucleoids. Cells with higher levels of FtsZ–GFP or with FtsA–GFP plus excess wild-type FtsZ were inhibited for cell division and often exhibited bright fluorescent spiral tubules that spanned the length of the filamentous cells. This suggests that FtsZ may switch from a septation-competent localized ring to an unlocalized spiral under some conditions and that FtsA can bind to FtsZ in both conformations. FtsZ–GFP also formed nonproductive but localized aggregates at a higher concentration that could represent FtsZ nucleation sites. The general domain structure of FtsZ–GFP resembles that of tubulin, since the C terminus of FtsZ is not required for polymerization but may regulate polymerization state. The N-terminal portion of Rhizobium FtsZ polymerized in Escherichia coli and appeared to copolymerize with E. coli FtsZ, suggesting a degree of interspecies functional conservation. Analysis of several deletions of FtsA–GFP suggests that multiple segments of FtsA are important for its localization to the FtsZ ring.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): Characterization of connective tissue growth factor as a member of the IGFBP superfamily

The insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine settings. Additionally, some IGFBPs appear to exhibit biological effects that are IGF independent. The six high-affinity IGFBPs that have been characterized to date exhibit 40–60% amino acid sequence identity overall, with the most conserved sequences in their NH2 and COOH termini. We have recently demonstrated that the product of the mac25/IGFBP-7 gene, which shows significant conservation in the NH2 terminus, including an “IGFBP motif” (GCGCCXXC), exhibits low-affinity IGF binding. The closely related mammalian genes connective tissue growth factor (CTGF) gene, nov, and cyr61 encode secreted proteins that also contain the conserved sequences and IGFBP motifs in their NH2 termini. To ascertain if these genes, along with mac25/IGFBP-7, encode a family of low-affinity IGFBPs, we assessed the IGF binding characteristics of recombinant human CTGF (rhCTGF). The ability of baculovirus-synthesized rhCTGF to bind IGFs was demonstrated by Western ligand blotting, affinity cross-linking, and competitive affinity binding assays using 125I-labeled IGF-I or IGF-II and unlabeled IGFs. CTGF, like mac25/IGFBP-7, specifically binds IGFs, although with relatively low affinity. On the basis of these data, we propose that CTGF represents another member of the IGFBP family (IGFBP-8) and that the CTGF gene, mac25/IGFBP-7, nov, and cyr61 are members of a family of low-affinity IGFBP genes. These genes, along with those encoding the high-affinity IGFBPs 1–6, together constitute an IGFBP superfamily whose products function in IGF-dependent or IGF-independent modes to regulate normal and neoplastic cell growth.

CooB plays a chaperone-like role for the proteins involved in formation of CS1 pili of enterotoxigenic Escherichia coli

CS1 pili serve as the prototype of a class of filamentous appendages found on the surface of strains of enterotoxigenic Escherichia coli. The four genes needed to synthesize functional CS1 pili in E. coli K12 are: cooA, which encodes the major pilin protein; cooD, which encodes a minor pilin protein found at the tip of the structure; cooC, which encodes a protein found in the outer membrane of piliated bacteria; and cooB. We show here that CooB, which is required for pilus assembly but is not part of the final structure, stabilizes CooA, CooC, and CooD. We previously reported that CooB is complexed with CooA in the periplasm and show here that CooB also is found complexed with CooD in the periplasm. CooB is associated with the membrane fraction only in the presence of CooC, suggesting that these two proteins also interact. This suggests that although it has no homology to known chaperone proteins, CooB serves a chaperone-like role for assembly of CS1.

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.