Metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF): selectivity and conformational consequences of histidine modification.

We have studied the metal-catalyzed oxidation (MCO) of brain-derived neurotrophic factor (BDNF) with regard to target sites and potential conformational changes of the protein. The exposure of BDNF to three different levels of ascorbate/Cu(II)/O2 [20 microM Cu(II), 2 mM ascorbate (level 1); 20 microM Cu(II), 4 mM ascorbate (level 2); 40 microM Cu(II), 4 mM ascorbate (level 3)], chosen based on the extent of chemical modification of Met and His, respectively, resulted in the exclusive oxidation of a buried Met residue, Met92, at level 1 but in the predominant oxidation of His at level 3. His modification had a significant impact on the structure of BDNF, as quantified by CD and ANSA fluorescence measurements, while Met oxidation had not, also assessed through complementary oxidation of BDNF through hydrogen peroxide. Our ultimate objective was the correlation of the surface exposure of an oxidized His residue in a protein with potential effects on the conformational integrity of the oxidized protein. In a series of three proteins, human growth hormone (hGH), human relaxin (hR1x), and BDNF, we have now observed that His oxidation is paralleled by significant conformational changes when the target His residue is more surface exposed (hR1x, BDNF) while conformational consequences of His modification are less significant when the target His residues are more buried in the interior of the protein (hGH).     

Surface plasmon resonance-based highly sensitive immunosensing for brain natriuretic peptide using nanobeads for signal amplification

Site-specific metal-catalyzed oxidation (MCO) was applied to characterize the metal-binding site (MBS) of recombinant human prolactin (hPRL), which belongs to the hematopoietic cytokine family. Copper and ascorbate of various concentrations were used to initiate the oxidation of hPRL, and the oxidation-sensitive motifs were characterized and quantitated by mass spectrometry. Based on the results obtained with 10 microM Cu(2+) and 0.3-2.0mM ascorbate, we propose that the MBS in hPRL is composed of His27, His30, and His173. This result shows the similarity of hPRL to human growth hormone (hGH), a member of the same family as hPRL, where the MBS is composed of His18, His21, and Glu174.     

Metal-catalyzed oxidation reactions and mass spectrometry: the roles of ascorbate and different oxidizing agents in determining Cu-protein-binding sites

Further study has been made of metal-catalyzed oxidation (MCO) reactions and mass spectrometry as a method to determine the binding site of copper in metalloproteins. The role of ascorbate and a variety of oxidizing agents, including O2, H2O2, and S2O8(2-), have been investigated using Cu/Zn superoxide dismutase (SOD) as a model system. Ascorbate is found to play two competing roles in the MCO reactions. It reduces Cu(II), which initiates and maintains the generation of reactive oxygen species, and it scavenges radicals, which helps to localize oxidation products to amino acids near the metal center. An ascorbate concentration of 100 mM is found to be optimal with regard to localizing oxidation products to only the Cu-binding residues (His44, His46, His61, and His118) of Cu/Zn SOD. This concentration of ascorbate is very similar to the optimum concentration found in our previous studies of different Cu-binding proteins. Another notable result from this study is the observation that S2O8(2-) is more effective as an oxidant than O2 or H2O2 in the MCO reactions. Because S2O8(2-) is more stable in solution than H2O2, using it as an oxidizing agent results in much less nonspecific oxidation to the protein. The overall results of this study suggest that general MCO reaction conditions may exist for determining the metal-binding site of a wide range of Cu-binding proteins.     

Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals

Oxidative stress is believed to play a central role in the pathogenesis of prion diseases, a group of fatal neurodegenerative disorders associated with a conformational change in the prion protein (PrP(C)). The precise physiological function of PrP(C) remains uncertain; however, Cu(2+) binds to PrP(C) in vivo, suggesting a role for PrP(C) in copper homeostasis. Here we examine the oxidative processes associated with PrP(C) and Cu(2+). (1)H NMR was used to monitor chemical modifications of PrP fragments. Incubation of PrP fragments with ascorbate and CuCl(2) showed specific metal-catalyzed oxidation of histidine residues, His(96/111), and the methionine residues, Met(109/112). The octarepeat region protects His(96/111) and Met(109/112) from oxidation, suggesting that PrP(90-231) might be more prone to chemical modification. We show that Cu(2+/+) redox cycling is not 'silenced' by Cu(2+) binding to PrP, as indicated by H(2)O(2) production for full-length PrP. Surprisingly, although detection of Cu(+) indicates that the octarepeat region of PrP is capable of reducing Cu(2+) even in the absence of ascorbate, H(2)O(2) is not generated unless ascorbate is present. Full-length PrP and fragments cause a dramatic reduction in detectable hydroxyl radicals in an ascorbate/Cu(2+)/O(2) system; however, levels of H(2)O(2) production are unaffected. This suggests that PrP does not affect levels of hydroxyl radical production via Fentons cycling, but the radicals cause highly localized chemical modification of PrP(C).     

Synthesis of bovine growth hormone in primates by using a herpesvirus vector.

A strain of herpesvirus saimiri containing a bovine growth hormone (bGH) gene under the control of the simian virus 40 (SV40) late-region promoter was constructed. This strain, bGH-Z20, was replication competent and stably harbored the bGH gene upon serial passage. Nonpermissive marmoset T cells persistently infected with bGH-Z20 produced a 0.9-kilobase RNA which contained all of the bGH exon sequences and appeared to initiate within the SV40 promoter region. However, in permissively infected owl monkey kidney cells, RNAs containing growth hormone sequences appeared to initiate from herpesvirus saimiri promoters positioned upstream from the SV40-growth hormone gene. Persistently infected T cells in culture secreted 500 ng of bGH protein per 10(6) cells per 24 h during the several months of testing. The secreted protein was 21 kilodaltons, the size of authentic bGH. New World primates experimentally infected with bGH-Z20 produced circulating bGH and developed immunoglobulin G antibodies directed against bGH. Because herpesviruses characteristically remain latent in the infected host, these observations suggest a means for replacing gene products missing or defective in hereditary genetic disorders.     

牛生长激素结构不均一性的研究

本文报导用常规方法分离纯化的牛生长激素,在还原性SDS-11％聚丙烯酰胺凝胶电泳中呈分子量很接近的两条主带(22KD,21.5KD)。用单克隆抗体和多克隆抗体亲和层析技术分析了不同分子量形式的牛生长激素的转化,结果表明:牛生长激素可能在pH5.5条件下转化为21.5KD分子,在pH8.3条件下则转化18KD分子。这几种形式的牛生长激素均保留与抗体的结合力,但亲和力不尽相同,如在亲和层析的洗脱性质上存在差异。已检验分离并部分纯化了18KD分子以备作进一步的研究。     

Redox regulation of copper-metallothionein

Copper (Cu) is an essential element whose localization within cells must be carefully controlled to avoid Cu-dependent redox cycling. Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects during metal exposure and oxidative stress. The specific role of MTs, however, in modulating Cu-dependent redox cycling remains unresolved. Our studies utilized a chemically defined model system to study MT modulation of Cu-dependent redox cycling under reducing (Cu/ascorbate) and mild oxidizing (Cu/ascorbate + H2O2) conditions. In the presence of Cu and ascorbate, MT blocked Cu-dependent lipid oxidation and ascorbyl radical formation with a stoichiometry corresponding to Cu/MT ratios 相似文献   

The artificial AT-rich block enhanced the production of bovine growth hormone in Escherichia coli

In order to increase the synthesis of bovine growth hormone (bGH) using T7 promoter system in E. coli, the artificial AT-rich block was introduced into the upstream region of a consensus Shine-Dalgarno (SD) sequence and the spacer region (between SD and ATG codon) was enriched with A and T nucleotides. The cells harboring pTAJ plasmids with AT-rich block produced bGH in the range of 3% to 25% and the cells harboring pTBJ plasmids with AT-rich sequence in the spacer region from 0.8% to 20% of total cell proteins. This result suggests that AT rich block and AT nucleotides in the spacer region destabilize mRNA secondary structure, depending on the downstream coding information of bGH gene and also, implying that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.     

Fe2+-catalyzed site-specific cleavage of the large subunit of ribulose 1,5-bisphosphate carboxylase close to the active site

Previous work has demonstrated that the large subunit (rbcL) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from wheat is cleaved at Gly-329 by the Fe(2+)/ascorbate/H(2)O(2) system (Ishida, H., Makino, A., and Mae, T. (1999) J. Biol. Chem. 274, 5222-5226). In this study, we found that the rbcL could also be cleaved into several other fragments by increasing the incubation time or the Fe(2+) concentration. By combining immunoblotting with N-terminal amino acid sequencing, cleavage sites were identified at Gly-404, Gly-380, Gly-329, Ala-296, Asp-203, and Gly-122. Conformational analysis demonstrated that five of them are located in the alpha/beta-barrel, whereas Gly-122 is in the N-terminal domain but near the bound metal in the adjacent rbcL. All of these residues are at or very close to the active site and are just around the metal-binding site within a radius of 12 A. Furthermore, their C(alpha)H groups are completely or partially exposed to the bound metal. A radical scavenger, activation of RuBisCo, or binding of a reaction-intermediate analogue to the activated RuBisCo, inhibited the fragmentation. These results strongly suggest that the rbcL is cleaved by reactive oxygen species generated at the metal-binding site and that proximity and favorable orientation are probably the most important parameters in determining the cleavage sites.     

Enhancement of bovine growth hormone gene expression by increasing the plasmid copy number

Effect of copy number on the expression of bovine growth hormone gene (bGH) was investigated using the copy number mutants such as pKBJ10, pBJ( tet)10, pUBJ10-1, and pUBJ10 plasmids. The cells harboring plasmids below 84 copies/cell did not produced detectable levels of bGH. When the ColE1 replicon was replaced with the mutated ColE1 replicon originated from pUC19 plasmid, the copy number was increased to about 300 copies/cell and bGH production was enhanced by 11.5% (pUBJ10-1) and 12.3% of total cell protein (pUBJ10). A large amount of mRNA caused by increment of copy number would be needed to overcome some inhibitory threshold and might be an important factor for regulating bGH expression.     

An MspI polymorphism at the bovine growth hormone (bGH) gene is linked to a locus affecting milk protein percentage

SSCP analysis of the bovine growth hormone (bGH) gene in Israel Holstein dairy cattle uncovered five intragenic haplotypes, denoted A to E. Of these, Haplotype E differed from the others at six fragments; one of which corresponded to the polymorphic MspI site in intron III, at which haplotype E carried the disabled MspI (-) allele. Haplotype E was observed in a single sire only, carrying haplotype A as the second bGH allele. In 523 daughters of this sire genotyped for the MspI polymorphism, heterozygous (+/-) as compared to homozygous (+/+) daughters, showed a significant increasing effect on protein percentage and kg protein per year; and a decreasing effect (P < 0.10) on milk somatic cell counts (MSSC). None of the daughters were homozygous (-/-), indicating that the frequency of this allele in the general population was essentially zero. Calculated skewness (g1) values for the two daughter groups differed significantly with (+/-) daughters showing negative skewness (in the direction of lower protein percentage), and (+/+) daughters positive skewness (in the direction of higher protein percentage). The direction of skewness in each group is indicative of the presence of a QTL having an increasing effect on milk protein percentage in coupling linkage with the MspI (-) allele in this sire, but at some distance from it. Maximum likelihood estimates of the proportion of recombination (r) between the putative QTL and bGH, and the allele substitution effect at the QTL (d), were r = 0.33, a = 0.07% protein, with standard errors 0.058 and 0.009% protein, respectively.     

Restriction fragment length polymorphisms associated with growth hormone and prolactin genes in Holstein bulls: evidence for a novel growth hormone allele

Summary. Sperm DNA isolated from sons of three extensively used US Holstein bulls was screened for differences associated with the primary gene structure of the bovine growth hormone (bGH) and prolactin (bPrl) genes. Southern blot analysis of DNA digested with 10 restriction enzymes revealed that offspring from two of the three bull families exhibited polymorphisms around the bGH and bPrl genes. Restriction fragment length polymorphisms (RFLPs) around the bGH gene were detected with five enzymes, whereas three enzymes revealed RFLPs around the bPrl gene. At least three structural differences were predicted around the bGH gene. The most common variant hybridization pattern appeared to involve an insertion/deletion located downstream of the conserved 3' Eco RI site. The presence of RFLPs in the genes coding for these pituitary hormones within a familial line may provide the basis for genetic markers associated with lactation and mammary development.     

Serum leptin responses after acute resistance exercise protocols.

This study examined the acute effects of maximum strength (MS), muscular hypertrophy (MH), and strength endurance (SE) resistance exercise protocols on serum leptin. Ten young lean men (age = 23 +/- 4 yr; body weight = 79.6 +/- 5.2 kg; body fat = 10.2 +/- 3.9%) participated in MS [4 sets x 5 repetitions (reps) at 88% of 1 repetition maximum (1 RM) with 3 min of rest between sets], MH (4 sets x 10 reps at 75% of 1 RM with 2 min of rest between sets), SE (4 sets x 15 reps at 60% of 1 RM with 1 min of rest between sets), and control (C) sessions. Blood samples were collected before and immediately after exercise and after 30 min of recovery. Serum leptin at 30 min of recovery exhibited similar reductions from baseline after the MS (-20 +/- 5%), MH (-20 +/- 4%), and SE (-15 +/- 6%) protocols that were comparable to fasting-induced reduction in the C session (-12 +/- 3%) (P < 0.05). Furthermore, no differences were found in serum leptin among the MS, MH, SE, and C sessions immediately after exercise and at 30 min of recovery (P > 0.05). Cortisol was higher (P < 0.05) after the MH and SE protocols than after the MS and C sessions. Glucose and growth hormone were higher (P < 0.05) after exercise in the MS, MH, and SE protocols than after the C session. In conclusion, typical resistance exercise protocols designed for development of MS, MH, and SE did not result in serum leptin changes when sampled immediately or 30 min postexercise.     

Skeletal muscle afferent regulation of bioassayable growth hormone in the rat pituitary

There are forms of growth hormone (GH) in theplasma and pituitary of the rat and in the plasma of humans that areundetected by presently available immunoassays (iGH) but can bemeasured by bioassay (bGH). Although the regulation of iGHrelease is well documented, the mechanism(s) of bGH release is unclear.On the basis of changes in bGH and iGH secretion in rats that had been exposed to microgravity conditions, we hypothesized that neural afferents play a role in regulating the release of these hormones. Toexamine whether bGH secretion can be modulated by afferent input fromskeletal muscle, the proximal or distal ends of severed hindlimb fastmuscle nerves were stimulated (~2 times threshold) in anesthetizedrats. Plasma bGH increased ~250%, and pituitary bGH decreased~60% after proximal nerve trunk stimulation. The bGH response wasindependent of muscle mass or whether the muscles were flexors orextensors. Distal nerve stimulation had little or no effect on plasmaor pituitary bGH. Plasma iGH concentrations were unchanged afterproximal nerve stimulation. Although there may be multiple regulatorymechanisms of bGH, the present results demonstrate that the activationof low-threshold afferents from fast skeletal muscles can play aregulatory role in the release of bGH, but not iGH, from the pituitaryin anesthetized rats.

     

Binding and signalling properties of a growth hormone enhancing monoclonal antibody

We have used a sequential, qualitative biosensor based assay to demonstrate that OA15, a monoclonal antibody which enhances in vivo the activity of bovine growth hormone (bGH) does not disrupt the interaction between bGH and its cognate receptor (as represented by recombinant bovine GH binding protein -rbGHBP). We have confirmed this using a classical cell-based radio-receptor assay with the GH-responsive mouse pre-adipocyte cell line 3T3-F442A. The fact that OA15 binding to bGH still allows hormone to interact with its receptor, allows us to test the hypothesis that there is any amplification of signalling events following hormone-MAb treatment of 3T3-F442A cells. We have used as a reporter of GH activity the rapid stimulation of JAK-2 tyrosine phosphorylation which is a critical first step in GH signalling events. We demonstrate that binding of rbGH by OA15 attenuates hormone stimulation of JAK-2 tyrosine phosphorylation. We conclude that although OA15 does not disrupt GH-GH receptor (GHR) interactions it does interfere with subsequent GH activity at the molecular and cellular level. We further speculate therefore that the biological enhancing activity of this antibody is most likely due to an in vivo effect as presentation of antibody-hormone complexes to a GH-target cell inhibits hormone activity.     

Prolactin and Growth Hormone Binding in Mammary and Liver Tissue of Lactating Cows

Abstract

A radioligand/receptor binding assay was developed using homologous hormones to distinguish between bovine growth hormone (bGH) and bovine prolactin (bPRL) receptors in liver and mammary tissue of lactating cows. Mammary and liver tissues were homogenized in 0.3 M sucrose and centrifuged at 100,000 x g over a 1.3 M sucrose density gradient. Membranes from the 0.3 - 1.3 M sucrose interface were incubated with 1 ng of iodinated bGH or bPRL for 20 h at 22°C in the presence of increasing concentrations of native bGH or bPRL. High affinity receptor binding sites were found for bPRL in liver and mammary tissue membranes (Ka=3.2 and 1.3 × 10⁸ 1/mol with 34 and 63 fmol receptors/mg liver and mammary membrane protein, respectively) and for bGH only in liver tissue (Ka=1.8 × 10⁹ 1/mol, 18 fmol receptors/mg membrane protein). Receptor number estimates were 3 and 11 times higher in mammary and liver tissue using a heterologous hGH system indicating that heterologous systems may overestimate the number of receptors in bovine tissue. The absence of demonstratable bGH receptors in lactating bovine mammary tissue supports in vitro results of others with isolated mammary tissue indicating that the positive effect of bGH on milk production in intact cows is via an indirect mechanism.     

Influence of bovine growth hormone on the growth dynamics of mosaic muscle in relation to somatic growth of rainbow trout, Salmo gairdneri Richardson

Bovine growth hormone (bGH), administered intramuscularly at 20 μg^-1 every 2 weeks, produced faster somatic growth in rainbow trout fingerlings to approximately 30 cm than in those not receiving the hormone. Both groups were maintained at 12° C, with a photoperiod of 16 : 8 LD, and on ad libitum rations daily. The faster growth was characterized by a controlled increase in input of fibres into the mosaic muscle (which dominates the body tissues by its bulk). It is not known if the enhanced input of fibres is an example of the known ability of the mosaic fibre mass to respond with flexibility and precision to widely differing somatic growth rates, or is a specific consequence of the growth hormone. In other fingerling trout receiving 4-5% daily rations and consequently growing slowly, bGH did not stimulate growth or evidently modify fibre input dynamics. Body dry weight, and condition ( K ), though modified by rations and growth rate, evidently had little effect on muscle fibre growth.     

双抗体免疫沉淀法纯化牛生长激素poly(A)~+RNA的研究

我们用双抗体免疫沉淀法,从牛垂体多聚核糖体中分离出牛生长激素特异多聚核糖体,由此多聚核糖体纯化的牛生长激素Poly(A)~+RNA,可以在麦胚体外翻译系统中和兔网织红细胞体外翻译系统中促进~(14)C-亮氨酸的参入。合成的含~(14)-亮氨酸的翻译产物中有91％可以被牛生长激素抗体沉淀。用SDS-11％PAGE对翻译产物进行鉴定表明,翻译产物在25KD处呈一条放射自显影带,与报导的牛生长激素前体分子量相吻合。     

Monoclonal antibodies against bovine growth hormone.

A number of mouse x mouse hybridomas producing monoclonal antibodies (MAbs) against bovine growth hormone (bGH) were prepared by fusion of spleen cells from bGH-primed mice (Balb/c) with non-secretory mouse myeloma cells (PAIOP3) and characterized. MAbs obtained from three fusion experiments belonged to IgM, IgG1 and IgG2b class/subclass of antibodies. Cross-reaction studies indicated that generated antibodies were against three different epitopes of bGH. VIA6E8 (IgG1) and VIIB2E11C9 (IgM) did not cross-react with ovine prolactin (oPRL), ovine leutinizing hormone (oLH) and porcine follicle stimulating hormone. Antibody VIB3C9E8 (IgM) exhibited cross-reaction with oPRL and oLH. Antibody VIC1F9 (IgG2b) cross reacted with oPRL. All MAbs were against conformational epitopes of bGH.