Availability of folate bound to folate-binding protein: Environmental effects

The effect of FBP on folate bio-availability depends on its environment. The FBP of whole WPC enhances bioavailability of folates more than does purified FBP and its efficacy might be even greater when lipids are removed from the WPC. FBP polymerises and folate release from the polymer is found to be slower than that from the monomer. FBP has a role also as a folate receptor at cell surfaces and in this role folate binding increases polymerisation of FBP attached to lipid membranes.

Quantitative analysis of the time courses of enzyme-catalyzed reactions

The catalytic properties of enzymes are usually evaluated by measuring and analyzing reaction rates. However, analyzing the complete time course can be advantageous because it contains additional information about the properties of the enzyme. Moreover, for systems that are not at steady state, the analysis of time courses is the preferred method. One of the major barriers to the wide application of time courses is that it may be computationally more difficult to extract information from these experiments. Here the basic approach to analyzing time courses is described, together with some examples of the essential computer code to implement these analyses. A general method that can be applied to both steady state and non-steady-state systems is recommended. (C) 2001 academic Press.

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains

Monocyte macrophages (M phi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to M phi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca2+/Mg2+ enhanced DV binding. This argues against involvement of beta (2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains. (C) 2001 Elsevier Science B.V. All rights reserved.

Growth hormone binding protein in normal and aneuploid pregnancy: a paradoxical decrease in trisomy 18

Objective To explore whether abnormalities in growth hormone binding protein (GHBP) may underlie the growth restriction associated with fetal aneuploidy. Design A retrospective casecontrol study. Setting Monash Medical Centre, Clayton, Victoria, Australia. Population Twenty-one trisomy 18, and 30 trisomy 21 pregnancies, and 170 chromosomally normal pregnancies at 15-18 weeks of gestation representing three to five controls per case matched for source, gestation and duration of storage. Methods GHBP was measured using a ligand immunofunctional assay. Results In the chromosomally normal pregnancies GHBP levels decreased slightly but significantly across the narrow gestational window studied. Compared with controls, levels of GHBP, expressed as median (95% CI) multiples of the median (MoM), in the trisomy 21 pregnancies were similar, 1.0 (0.92-1.39) MoM and 1.27 (1.04-1.50) MoM, respectively; P = 0.061 (Mann-Whitney CI test) but were significantly reduced in the trisomy 18 pregnancies, 0.68 (0.51-0.84) MoM; P = 0.0014 (Mann-Whitney U test). Conclusions These data suggest that decreased levels of maternal growth hormone binding protein, and by implication growth hormone receptor complement, may underlie the early severe growth restriction that is characteristic of trisomy 18.

The human temporal cortex: Characterization of neurons expressing nitric oxide synthase, neuropeptides and calcium-binding proteins, and their glutamate receptor subunit profiles

Immunocytochemical techniques were used to examine the distribution of neurons immunoreactive (-ir) for nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) and calretinin (CH), in the inferotemporal gyros (Brodmann's area 21) of the human neocortex. Neurons that colocalized either nNOS or SOM with PV, CB or CR were also identified by double-labeling techniques. Furthermore, glutamate receptor subunit profiles (GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1) were also determined for these cells. The number and distribution of cells containing nNOS, SOM, NPY, PV, CB or CR differed for each antigen. In addition, distinct subpopulations of neurons displayed different degrees of colocalization of these antigens depending on which antigens were compared. Moreover, cells that contained nNOS, SOM, NPY, PV, GB or CR expressed different receptor subunit profiles. These results show that specific subpopulations of neurochemically identified nonpyramidal cells may be activated via different receptor subtypes. As these different subpopulations of cells project to specific regions of pyramidal calls, facilitation of subsets of these cells via different receptor subunits may activate different inhibitory circuits. Thus, various distinct, but overlapping, inhibitory circuits may act in concert in the modulation of normal cortical function, plasticity and disease.

The chemistry and biological effects of malondialdehyde-acetaldehyde adducts

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Geoffrey M. Thiele and Simon Worrall. The presentations were (1) The chemistry of malondialdehyde-acetaldehyde (MAA) adducts, by Dean J. Tuma; (2) The formation and clearance of MAA adducts in ethanol-fed rats, by Simon Worrall; (3) Immune responses to MAA adducts may play a role in the development of alcoholic liver disease, by Lynell W. Klassen; (4) Unique biological responses to MAA-modifled proteins that may play a role in the development and/or progression of alcoholic liver disease, by Geoffrey M. Thiele; (5) MAA-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells, by Todd A. Wyatt; and (6) An enzyme immune assay for serum antiacetaldehyde adduct antibody using low-density lipoprotein-adduct and its significance in alcoholic liver injury and ALDH2 heterozygotes, by Naruhiko Nagata.

Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells

Skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of the MyoD family and the MADS protein, MEF2, which function in a cooperative manner. The steroid receptor coactivator SRC-2/GRIP-1/TIF-2, is necessary for skeletal muscle differentiation, and functions as a cofactor for the transcription factor, MEF2. SRC-P belongs to the SRC family of transcriptional coactivators/cofactors that also includes SRC-1 and SRC-3/RAC-3/ACTR/ AIB-1. In this study we demonstrate that SRC-P is essentially localized in the nucleus of proliferating myoblasts; however, weak (but notable) expression is observed in the cytoplasm. Differentiation induces a predominant localization of SRC-P to the nucleus; furthermore, the nuclear staining is progressively more localized to dot-like structures or nuclear bodies. MEF2 is primarily expressed in the nucleus, although we observed a mosaic or variegated expression pattern in myoblasts; however, in myotubes all nuclei express MEF2. GRIP-1 and MEF2 are coexpressed in the nucleus during skeletal muscle differentiation, consistent with the direct interaction of these proteins. Rhabdomyosarcoma (RMS) cells derived from malignant skeletal muscle tumors have been proposed to be deficient in cofactors. Alveolar RMS cells very weakly express the steroid receptor coactivator, SRC-P, in a diffuse nucleocytoplasmic staining pattern. MEF2 and the cofactors, SRC-1 and SRC-3 are abundantly expressed in alveolar and embryonal RMS cells; however, the staining is not localized to the nucleus. Furthermore, the subcellular localization and transcriptional activity of MEF2C and a MEF2-dependent reporter are compromised in alveolar RMS cells. In contrast, embryonal RMS cells express SRC-2 in the nucleus, and MEF2 shuttles from the cytoplasm to the nucleus after serum withdrawal. In conclusion, this study suggests that the steroid receptor coactivator SRC-P and MEF2 are localized to the nucleus during the differentiation process. In contrast, RMS cells display aberrant transcription factor SRC localization and expression, which may underlie certain features of the RMS phenotype.

Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation

Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases, Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer, The monomeric unit of the protein was chemically synthesized in three parts (positions 1-51, 54-99, and 102-127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.

FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta -catenin and A-F-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure Of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta -catenin such that very little of either protein remained following 72 h culture. The residual beta -catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta -catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta -catenin and AF-6 in vivo. (C) 2001 Elsevier Science Ireland Lid. All rights reserved.

Familial corticosteroid-binding globulin deficiency due to a noval null mutation: Association with fatigue and relative hypotension

Corticosteroid-binding globulin is a 383-amino acid glycoprotein that serves a hormone transport role and may have functions related to the stress response and inflammation. We describe a 39-member Italian-Australian family with a novel complete loss of function (null) mutation of the corticosteroid-binding globulin gene. A second, previously described, mutation (Lyon) segregated independently in the same kindred. The novel exon 2 mutation led to a premature termination codon corresponding to residue -12 of the procorticosteroid-binding globulin molecule (c.121G->A). Among 32 family members there were 3 null homozygotes, 19 null heterozygotes, 2 compound heterozygotes, 3 Lyon heterozygotes, and 5 individuals without corticosteroid-binding globulin mutations. Plasma immunoreactive corticosteroid-binding globulin was undetectable in null homozygotes, and mean corticosteroid-binding globulin levels were reduced by approximately 50% at 18.7 ± 1.3 µg/ml (reference range, 30–52 µg/ml) in null heterozygotes. Morning total plasma cortisol levels were less than 1.8 µg/dl in homozygotes and were positively correlated to the plasma corticosteroid-binding globulin level in heterozygotes. Homozygotes and heterozygote null mutation subjects had a high prevalence of hypotension and fatigue. Among 19 adults with the null mutation, the systolic blood pressure z-score was 12.1 ± 3.5; 11 of 19 subjects (54%) had a systolic blood pressure below the third percentile. The mean diastolic blood pressure z-score was 18.1 ± 3.4; 8 of 19 subjects (42%) had a diastolic blood pressure z-score below 10. Idiopathic chronic fatigue was present in 12 of 14 adult null heterozygote subjects (86%) and in 2 of 3 null homozygotes. Five cases met the Centers for Disease Control criteria for chronic fatigue syndrome. Fatigue questionnaires revealed scores of 25.1 ± 2.5 in 18 adults with the mutation vs. 4.2 ± 1.5 in 23 healthy controls (P < 0.0001). Compound heterozygosity for both mutations resulted in plasma cortisol levels comparable to those in null homozygotes. Abnormal corticosteroid-binding globulin concentrations or binding affinity may lead to the misdiagnosis of isolated ACTH deficiency. The mechanism of the association between fatigue and relative hypotension is not established by these studies. As idiopathic fatigue disorders are associated with relatively low plasma cortisol, abnormalities of corticosteroid-binding globulin may be pathogenic.

Getting the adrenaline going: Crystal structure of the adrenaline-synthesizing enzyme PNMT

Background: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. Results: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 Angstrom. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. Conclusions: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.

Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis

Background & Aims: There is a significant relationship between inheritance of high transforming growth factor (TGF)-beta1 and angiotensinogen-producing genotypes and the development of progressive hepatic fibrosis in patients with chronic hepatitis C. In cardiac and renal fibrosis, TGF-beta1 production may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. The aim of the present study was to determine the effects of the angiotensin converting enzyme inhibitor, captopril, on the progression of hepatic fibrosis in the rat bile duct ligation model. Methods: Rats were treated with captopril (100 mg kg(-1) day(-1)) commencing 1 or 2 weeks after bile duct ligation. Animals with bile duct ligation only and sham-operated animals sewed as controls. Four weeks after bile duct ligation, indices of fibrosis were assessed. Results: Cap topril treatment significantly reduced hepatic hydroxyproline levels, mean fibrosis score, steady state messenger RNA levels of TGF-beta1 and procollagen alpha1(I), and matrix metalloproteinase 2 and 9 activity. Conclusions: Captopril significantly attenuates the progression of hepatic fibrosis in the vat bile duct ligation model, and its effectiveness should be studied in human chronic liver diseases associated with progressive fibrosis.

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.