Predictability of sequence homologies among lysine-rich histones by immunological distance

The relationship between immunological distance (I.D.) measured by microcomplement fixation and amino acid sequence difference for lysine-rich histones was tested using antisera to lysine-rich histones of known sequence, chicken H1 and H5, goose H5, and trout H1 as well as to trout H5. The best relationship between I.D. (y) and percent sequence difference (x) for lysine-rich histones, y = 2x, applies as well to other histones of known sequence but it differs from y = 5x, reported for other proteins and often used for histones. Although deviations indicate that I.D. is a poor predictor of primary sequence differences among histones, it suggests that trout H5 is more closely related to H1 than to chicken H5.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the in vitro release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all in vitro experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

缺失ORF73或ORF214基因对重组鸡痘病毒免疫应答的影响

【目的】旨在研究鸡痘病毒ORF73和ORF214编码蛋白是否具有IL-18结合蛋白的功能,以及ORF73或ORF214基因缺失后对重组病毒诱导免疫应答的影响。【方法】以缺失ORF73或ORF214基因并表达H5亚型AIVHA基因的重组鸡痘病毒(rFPVLP-△73LRH5A、rFPVLP-△214LRH5A)作为研究对象,以未缺失ORF73或ORF214基因而表达H5亚型AIVHA基因的重组鸡痘病毒(rFPVLP-12LSH5A)作为对照,检测重组病毒体外诱导SPF鸡脾细胞和外周血淋巴细胞产生IFN情况,同时检测重组病毒免疫SPF鸡后诱导的体液免疫、CD4+/CD8+比值、外周血淋巴细胞的增殖能力和H5亚型AIV强毒攻击后的免疫保护效力。【结果】rFPVLP-△73LRH5A和rFPVLP-△214LRH5A体外诱导脾细胞产生的IFN量显著高于rFPVLP-12LSH5A,而免疫10d后的CD4+/CD8+比值显著低于rFPVLP-12LSH5A;3种重组鸡痘病毒诱导外周血淋巴细胞增殖的能力没有明显差异;3种重组病毒在SPF鸡均产生针对H5亚型AIV的HI抗体,免疫14d后rFPVLP-△214LRH5A组诱生的HI抗体水平显著低于rFPVLP-12LSH5A组的抗体,但3组在免疫21d后HPAIV的致死性攻击时,均100%被保护。【结论】鸡痘病毒ORF73和ORF214编码蛋白具有IL-18结合蛋白抑制IFN产生的功能,虽然缺失株和亲本株重组鸡痘病毒在细胞和体液免疫应答存在一定差异,但在SPF鸡均能诱导产生良好的免疫保护。     

Monoclonal antibodies against distinct determinants of histone H5 bind to chromatin

A series of monoclonal antibodies specific for distinguishable epitopes in chromosomal protein histone H5 were obtained from mice immunized with either free H5 or H5 . RNA complexes. The antibodies elicited by H5 could be distinguished from those elicited by H5 . RNA by their binding to native or acid-denatured H5, by their interaction with the globular region of H5, and by their cross-reactivity with H1o. The specificity of the antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting experiments. The antibodies could distinguish between H5 and the closely related histones H1 and H1o. The binding of some of the antibodies to the antigens was dependent on the type of assay used, suggesting nonrandom binding of the antigen to the solid supports used in ELISA and immunoblotting. Competitive ELISA experiments indicate that 8 of the 11 antibodies characterized bind to distinct epitopes. Three monoclonal antibodies bind to epitopes which are in close spatial proximity, causing mutual steric hindrance. The monoclonal antibodies bind to nuclei of fixed cells and to isolated chromatin, indicating that the epitopes are present both in the purified protein and in chromatin-complexed H5. These monoclonal antibodies can be used to study the organization of distinct regions of histones H5 and H1o in chromatin and chromosomes.     

Role of hypothalamic serotonergic receptors in the control of lordosis behavior in the female rat

The role of serotonin in mediating hypothalamic control of sexual behavior in estrone-primed ovariectomized (OVX) rats was studied by comparing the lordotic patterns following medial preoptic (MPOA) and arcuate-ventromedial (ARC-VM) infusions of serotonin (5-HT), methysergide (MS), and vehicle. In the initial experiments, low receptivity (preinfusion receptivity: mean lordosis/mount ratio = 0.164) was maintained by priming each animal with a low dose of estrone 48 hr prior to mating. The infusion of MS in either the MPOA or ARC-VM area resulted in a significant enhancement of lordotic behavior from initial low receptivity, 5-HT infusions were found to have no statistically significant effect upon lordotic behavior. In order to corroborate the findings observed in the low preinfusion receptivity protocol, OVX rats were primed with higher doses of estrone to maintain a high level of receptivity (preinfusion receptivity: mean lordosis/mount ratio = 0.787). Using this protocol, significant depressions in lordotic behavior were observed following MPOA or ARC-VM infusions of 5-HT, It was thus proposed that serotonergic receptors within the MPOA or ARC-VM areas have inhibitory effects upon lordotic behavior. In addition to the effects of 5-HT upon estrogen-induced sexual receptivity, serotonergic influences upon luteinizing hormone-releasing hormone (LRH)-facilitated mating behavior were also evaluated. Comparisons were made between the lordotic responses following MPOA or ARC-VM infusions of vehicle, LRH, or LRH with 5-HT in OVX rats primed with low doses of estrone. The infusion of LRH into the MPOA or ARC-VM significantly enhanced lordotic behavior above vehicle levels. However, the addition of 5-HT to the LRH infusate abolished this behavioral enhancement. These findings indicated that LRH and 5-HT have opposing effects within forebrain areas known to be important for the control of lordotic behavior.     

Presence of histone H1o-related fraction in chicken liver

The lysine-rich histones of chicken liver were studied in order to see whether a protein similar to mammalian histone H1o was present in this lower vertebrate. The following biochemical methods were used: sodium dodecylsulphate and acid-urea electrophoresis, gel exclusion chromatography on BioGel P100, and ion-exchange chromatography on BioRex 70. Specific polyclonal antibodies were elicited against purified mouse liver H1o and chicken erythrocyte H5, and applied for the further characterization of the chicken H1 subfractions obtained chromatographically. The results from microcomplement fixation and enzyme-linked immunosorbent assays showed that the presumptive chicken liver H1o shared common antigenic determinants with the mammalian H1o and the chicken liver H5. Based on the combined biochemical and immunological evidence, we conclude that an H1o-like protein is present in quiescent differentiated avian cells. The data of Smith et al. [34], who did not find this specific lysine-rich histone in resting chicken cells, are discussed.     

Comparison of various immunological methods for distinguishing among mammalian pancreatic ribonucleases of known amino acid sequence

Summary Fourteen mammalian pancreatic ribonucleases of known amino acid sequence were compared by 1 or more of 3 different immunological methods: standard quantitative micro-complement fixation, spot-plate micro-complement fixation, and inhibition of phage inactivation. It was found that, while the results obtained by the 3 techniques were correlated with one another, the standard microcomplement fixation procedure was most versatile, economical of materials, and easiest to execute. The standard MCF technique was more sensitive than the spotplate technique to differences in amino acid sequence. The inhibition of phage inactivation method was more sensitive than the standard method for measuring differences among closely related RNases but proved impractical for amino acid differences over 15%; the MCF method could be extended to at least 30% sequence differences. The standard method, moreover, readily detected the single amino acid difference between dromedary and camel RNases.A linear relationship was found between immunological distance (y) in the MCF test and percent sequence difference (x) which fit the equationy=7x. The strength of the correlation between immunological distance and percent sequence difference is consistent with the proposal that a large fraction of the evolutionary substitutions of amino acids in ribonuclease are immunologically detectable. This could be explained either by a multideterminant hypothesis or by a pauci-determinant hypothesis which says that substitutions occurring outside determinants produce small conformational changes influencing determinant reactivity.This work was supported in part by grants from the National Science Foundation (DEB74-11866A01) and the National Institutes of Health (GM-21509) to A.C.W. and a Fulbright-Hays grant to G.W.W. Part of this work was carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.). The followingabbreviations are used in this work: RNase=ribonuclease; MCF=micro-complement fixation.     

Presence of histone H1o-related fraction in chicken liver

Abstract. The lysine-rich histones of chicken liver were studied in order to see whether a protein similar to mammalian histone H1o was present in this lower vertebrate. The following biochemical methods were used: sodium dodecylsulphate and acid-urea electrophoresis, gel exclusion chromatography on BioGel P100, and ion-exchange chromatography on BioRex 70. Specific polyclonal antibodies were elicited against purified mouse liver Hlo and chicken erythrocyte H5, and applied for the further characterization of the chicken H1 subfractions obtained chromatographically. The results from microcomplement fixation and enzymelinked immunosorbent assays showed that the presumptive chicken liver Hlo shared common antigenic determinants with the mammalian H1o and the chicken liver H5. Based on the combined biochemical and immunological evidence, we conclude that an H1o-like protein is present in quiescent differentiated avian cells. The data of Smith et al. [34], who did not find this specific lysine-rich histone in resting chicken cells, are discussed.     

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5 end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.     

Species variability of N-terminal sequence of avian erythrocyte-specific histone H5

A comparison of the N-terminal amino acid sequence of H5 isolated from chicken, quail, duck, goose and pigeon shows considerable sequence variation. With the possible exception of the very lysine-rich histone H1, H5 variation is much more extensive than that reported previously for other histones. The observed sequence diversity is in agreement with the known taxonomic grouping of these birds.     

Changes in the sympathetic-adrenal system during stress in rats with high and low resistance to acute hypoxia

Quantitative luminescent microscopy was used to examine the sympathetic system of cardiac ventricles (SSCV) and the adrenal medulla (AM) of adult Wistar male rats high-resistant (HRH) and low-resistant (LRH) to acute hypoxia and exposed norepinephrine (NE) stress. The relative area of fluorescence adrenergic terminals (RAFAT) of the ventricles and AM catecholamine levels (CL) were shown to be equal in control HRH and LRH rats. The LRH rats displayed a two-phase SSCV response in the first 6 hours of NE stress. Their RAFAT rose an hour later and their RAFAT in the basal zone of the left ventricle insignificantly exceeded that of HRH rats, RAFAT in the latter being unchanged by that time. At hour 6, the heart RAFAT decreased as compared to 1-hour and control levels in LRH and HRH rats and became equal in the two groups. The AM CL in LRH and HRH rats remained unaltered within the whole period of the examination. Despite the profound differences in the resistance of HRH and LRH rats to hypoxia, the responsiveness of their sympathoadrenal system (SAS) to stress is rather homogeneous. With stress, the sympathetic link of SAS is more labile than the adrenal one.     

Purification,Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

Background

Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation ‘Rhi o 1’.

Method

The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies.

Results

The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross-stimulate histamine release from the effector cells sensitized with anti-Rhi o 1 serum IgE. This cross-reactivity was found to be mediated by a common mAb4C3 recognizable conformational epitope. Bioinformatic studies revealed high degree of structural resemblances between the 4C3 binding sites of both the allergens.

Conclusion/Significance

The present study reports for the first time anew fungal aspartic protease allergen designated as Rhi o 1, which triggers IgE-mediated sensitization leading to various allergic diseases. Here we have characterized the recombinant Rhi o 1 and its immunological features including cross-reactive epitope information that will facilitate the component-resolved diagnosis of mold allergy.     

Immunofractionation of chromatin regions associated with histone H1o

Two monoclonal antibodies, which were elicited against histone H5, bind to purified rat liver chromatin and to rat liver H1o but not to rat liver H1. The monoclonal antibodies were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to fractionate rat liver oligonucleosomes. Enzyme-linked immunoabsorbant assay (ELISA) and immunoblotting experiments indicate that the nucleosomes bound to the column were tenfold enriched in their content of H1o. Oligonucleosomes, prepared from the livers of either untreated or 3-methylcholanthrene-treated adult rats, were fractionated on the anti-H1o affinity column. The DNA purified from the unfractionated nucleosomes, from the unbound nucleosomes and from the nucleosomes which were bound to the column was examined with various 32P-labeled probes. A slight enrichment in H1o was detected in the coding region of the rat albumin gene. In contrast DNA which was bound to the column was significantly depleted in sequences hybridizing with total cellular RNA (which contains mostly ribosomal RNA) and with sequences hybridizing to the 3'-terminal region of a cytochrome P-450 gene, which is inducible by the chemical carcinogen 3-methylcholanthrene, regardless of whether isolated from control or from carcinogen-treated rat livers. Our experiments clearly demonstrate that chromatin can be efficiently immunofractionated. The results suggest that the H1o content of chromatin regions containing genes which are constitutively transcribed is not necessarily different from that of regions containing non-transcribed genes and that highly inducible genes may be segregated into chromatin regions which are depleted of H1o.     

禽流感病毒分离株NS基因同源性及等位基因类型分析

目的　克隆测定国内具有代表性的禽流感病毒 (AIV)的非结构 (NS)蛋白基因核苷酸序列 ,分析其同源性和等位基因类型 ,为进一步探索禽流感NS蛋白抗体监测方法奠定基础。方法　经RT PCR扩增了国内 3株H9N2、2株H5N1、2株H7N2亚型AIV分离株的NS蛋白基因 ,并把扩增的基因片段克隆到pGEM T载体中测序 ,将测序结果与GenBank中的核苷酸序列进行同源性比较 ,绘制基因进化树。结果　经测序获得了各AIV分离株NS基因的完整编码序列。同源性分析表明 ,3株H9亚型AIV的NS基因之间的同源性为 96 %～ 98% ;两株H5亚型AIVNS基因同源性为 91 6 % ;两株H7亚型AIV的NS基因同源性为 98 9%。H5和H9亚型分离株的NS基因之间的同源性均高于 90 % ;而H7N2亚型分离株与其它两种亚型分离株的NS基因同源性约为 6 0 %～ 70 %。在AIVNS基因系统发育进化树中 ,H5、H9亚型分离株都处于等位基因A群内 ;3株H9亚型分离株的进化关系较近 ,与香港、广东的部分H5N1病毒株起源相同 ,而 2株H5病毒的NS基因则处于不同分枝内 ;2株H7亚型分离株的NS基因都处于等位基因B群内 ,进化关系较近。结论　这 7株国内AIV分离株的NS基因之间的同源性差异较大 ,约为 6 0 %～ 99% ,且包括A、B两种类型的等位基因     

Differences between EcoRI nonspecific and "star" sequence complexes revealed by osmotic stress

The binding of the restriction endonuclease EcoRI to DNA is exceptionally specific. Even a single basepair change ("star" sequence) from the recognition sequence, GAATTC, decreases the binding free energy of EcoRI to values nearly indistinguishable from nonspecific binding. The difference in the number of waters sequestered by the protein-DNA complexes of the "star" sequences TAATTC and CAATTC and by the specific sequence complex determined from the dependence of binding free energy on water activity is also practically indistinguishable at low osmotic pressures from the 110 water molecules sequestered by nonspecific sequence complexes. Novel measurements of the dissociation rates of noncognate sequence complexes and competition equilibrium show that sequestered water can be removed from "star" sequence complexes by high osmotic pressure, but not from a nonspecific complex. By 5 Osm, the TAATTC "star" sequence complex has lost almost 90 of the approximately 110 waters initially present. It is more difficult to remove water from the CAATTC "star" sequence complex. The sequence dependence of water loss correlates with the known sequence dependence of "star" cleavage activity.     

Genome sequence analysis of H5N1 influenza a virus isolated from a vietnamese in 2007

Highly pathogenic H5N1 avian influenza A virus (AIV) crossed the species barrier and caused a number of deaths in humans in Vietnam and 14 other countries. Since the last report of human H5N1 infection in November 2005, the first documented H5N1 human infection was reported in June 2007 in Vietnam and was followed by 7 more cases, including 5 fatalities. In this study, we isolated and analyzed the full length of the H5N1 genome from a sample from the first patient in 2007. Phylogenetic analysis of eight genomic segments of the H5N1 virus strain (A/Vietnam/HN/2007, VNH07) revealed that this strain appears to be of genotype V and contains the HA gene, which is classified into clade 2.3.4. The deduced amino acid sequence of the HA protein has a typical affinity sequence for α2,3 linkage (SAα2,3-Gal) receptors and typical multibasic cleavage sequences. Compared with other H5N1 isolates, VNH07 showed that the possible reassortments for the NA and NP segments occurred between A/goose/Guangxi/3017/2005-like iso?lates (2.3.2) and A/human/Zhejiang/16/2006-like isolates (2.3.4).     

H 1 histone and histone variants in Trypanosoma cruzi

Trypanosoma cruzi chromatin is not condensed in chromosomes during mitosis. In previous studies a characteristic H 1 was not found in SDS or in acid-urea-PAGE. Consequently, it was proposed that the particular behavior of T. cruzi chromatin in dividing cells was due to the absence of an H 1 histone. In the present work, histones from this parasite were systematically characterized by spectrofluorometric analysis, amino acid composition, PAGE in one and in two dimensions, differential extraction with PCA and TCA, immunological cross-reactivity with antisera, and immunoblotting. We conclude that T. cruzi contains all five histones, H 1 presenting solubility and immunological properties similar to those in other species, but with a particular electrophoretic mobility in Triton-PAGE. Thus an explanation other than the absence of H 1 should be offered in order to understand the behavior of T. cruzi chromatin during mitosis. Moreover, histone variants were described by two-dimensional PAGE. The presence of histone variants suggests that they may participate in the regulation of cell proliferation and differentiation of this parasite, as it has been postulated for higher eukaryotes.