Differential effects of protein malnutrition and ascorbic acid deficiency on histidine metabolism in the brains of infant nonhuman primates

Abstract: Male infant nonhuman primates (M. nemes-trina) born in captivity were used in the study. They were divided into three groups. The first group of three animals was fed a 20% casein diet and the second group of six monkeys received a 2.0% casein diet. The third group of four monkeys received a 20% casein diet totally devoid of ascorbic acid for 3.5 weeks before the diet was supplemented with ascorbic acid (20 mg/kg diet). All the diets were given to the animals in two daily rations of 100 g/animal. The monkeys fed a 2% casein diet failed to grow, and after about 3.5 months showed variable degrees of edema, hypoalbuminemia, evidence of psychomotor disturbance, depressed plasma levels of many essential amino acids, and other features consistent with the diagnosis of protein-energy malnutrition. Examination of the brains revealed significant alterations in the levels of histidine (+ 172%) and homocarnosine (+ 146%) in comparison with the control well-fed monkeys. Associated with the increase in brain histidine was a marked elevation of brain histamine level. Protein deficiency also led to poor brain retention of ascorbic acid but not to the same degree observed in the ascorbic acid-deficient animals. The latter group of animals, after receiving their diet for about 8 months, demonstrated a modest elevation in the plasma levels of most amino acids in comparison with controls. Ascorbic acid deficiency elicited a significant reduction (p < 0.01) in brain level of histidine, with hardly any change in homocarnosine level. In addition, vitamin C deficiency produced elevation of brain histamine level comparable to findings in the protein-energy-deficient monkeys. The results suggested that protein deficiency raised brain histamine level mainly through increased availability of the precursor amino acid histidine, while defective degradation might account for the increased brain level of this amine in ascorbic acid-deficient monkeys. Histamine has been proposed to have a predominantly depressant action on relevant neurons, and has also been shown to participate with other neuro-transmitters in influencing the function of the pituitary gland by regulating release of the hypothalamic hormones into the portal vessels. The relevance of the findings of marked increases in brain histamine in experimental protein and ascorbic acid deficiencies to the behavioral and extensive endocrinological alterations seen in human malnutrition deserves some intensive investigation.     

24-hour variation in content and release of hypothalamic neuropeptides in the rat

The tissue content of up to eight neuropeptides, viz bombesin (BOM), cholecystokinin (CCK-8), neurotensin (NT), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), somatostatin (SRIF), substance P (SP) and vasoactive intestinal polypeptide (VIP), in rat hypothalami removed at various times of the day, was measured using specific radioimmunoassays. There was significant variation in the content of BOM, CCK-8, NT, PHI, SP and VIP across a 24-h period. The levels of BOM, CCK-8 and NT were lowest around the onset of darkness (1900 h) and rose throughout the night to reach a peak around the time of lights on. Hypothalamic content of all eight peptides fell between 0700 h and 1300 h by an average of 45 +/- 4%. Basal release of these peptides, as well as that in the presence of 48 mM potassium (K+), was measured from hypothalami removed between 0700 and 1900 h and incubated in vitro in a CSF-like medium. Basal secretion of NT significantly increased, whilst that of CCK-8 significantly decreased over the same period. There was no significant change in the basal release of the other neuropeptides. The release in the presence of 48 mM K+ of SP decreased significantly during the day, whilst that of VIP significantly increased. There was also a significant change in the stimulated release of BOM, levels falling during the morning and rising again at 1900 h. 48 mM K+ caused a significant increase in the release of SRIF and SP at all times tested. Whilst 48 mM K+ induced a significantly higher release of CCK-8 and NT in the morning, this stimulus was ineffective in the evening. The contrary was true in the case of BOM, NPY and VIP, where a significant stimulation was induced only at 1900 h. The possible implications of these findings are discussed.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

利用TMV—U1作为载体表达鼠肝炎病毒S糖蛋白片段

为了验证转基因烟草中表达的外壳蛋白（ＣＰ）能够重新包被侵入的烟草花叶病毒（ＴＭＶ）的假设,利用抗原表位标记的方法观察ＣＰ亚单位在病毒５′端的交换。通过ＰＣＲ 方法将来源于鼠肝炎病毒（ＭＨＶ） Ｓ蛋白的两个小肽段（１１ ａ．ａ．和１５ ａ．ａ．）的ＤＮＡ序列分别插入ＴＭＶ－Ｕ１ ＣＰ基因邻近３′端的两个位点,并构建了带有外源序列的ＴＭＶ 侵染克隆Ｖ９ （１１ ａ．ａ．）和Ｅ１５ （１５ ａ．ａ．）。通过体外转录反应,得到Ｖ９ ＲＮＡ 及Ｅ１５ ＲＮＡ。突变病毒ＲＮＡ 侵染烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）后表现不同特性。Ｖ９ 和Ｅ１５ 侵染ＸａｎｔｈｉＮＮ烟草后同野生型ＴＭＶ一样产生枯斑。但是,当它们侵染Ｘａｎｔｈｉｎｎ 烟草时,Ｖ９ 产生同侵染ＸａｎｔｈｉＮＮ 烟草相同的枯斑,而Ｅ１５的特性同ＴＭＶ－Ｕ１几乎完全相同,能对Ｘａｎｔｈｉｎｎ 烟草进行系统侵染并在叶片中聚集大量的带有外源片段的外壳蛋白,而且病毒的结构极其稳定。Ｖ９ 和Ｅ１５ 特性的差异可能是由于外源片段在外壳蛋白中存在位置的不同影响了外壳蛋白的结构所致     

The native state of apomyoglobin described by proton NMR spectroscopy: interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS.

Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His-64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His-82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481-6491). The packing of the side chains and the low pKa of His-64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter-amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.     

Response regulation in bacterial chemotaxis

The signal transduction system that mediates bacterial chemotaxis allows cells to moduate their swimming behavior in response to fluctuations in chemical stimuli. Receptors at the cell surface receive information from the surroundings. Signals are then passed from the receptors to cytoplasmic chemotaxis components: CheA, CheW, CheZ, CheR, and CheB. These proteins function to regulate the level of phosphorylation of a response regulator designated CheY that interacts with the flagellar motor switch complex to control swimming behavior. The structure of CheY has been determined. Magnesium ion is essential for activity. The active site contains highly conserved Asp residues that are required for divalent metal ion binding and CheY phosphorylation. Another residue-at the active site, Lys109, is important in the phosphorylation-induced conformational change that facilitates communication with the switch complex and another chemotaxis component, CheZ. CheZ facilitates the dephosphorylation of phospho-CheY. Defects in CheY and CheZ can be suppressed by mutations in the flagellar switch complex. CheZ is thought to modulate the switch bias by varying the level of phospho-CheY. © 1993 Wiley-Liss, Inc.     

SURVIVAL RATES FOR THE HAWAIIAN MONK SEAL (MONACHUS SCHAUINSLANDI)

Abstract: Endangered Hawaiian monk seal ( Monachs schauinslandi ) pups at all the major breeding islands in the Northwestern Hawaiian Islands have been tagged since the early 1980s. Pups were double flipper tagged as soon as possible post-weaning. With few exceptions, an extensive tag resighting effort was conducted annually at the same islands. These resighting data were used to estimate seal survival rates from the time of tagging to age one at all locations using the ratio of seals alive in the second year to number of pups tagged. These survival rates among the islands, from weaning to age one, averaged over the years of the study, ranged from 0.80 to 0.90. For young seals over age one, capture-recapture methods were used to calculate survival pooled through several years, and these rates ranged from 0.85 to 0.98. At French Frigate Shoals and Laysan Island, the higher numbers of tagged pups allowed separate estimates of male and female survival to be calculated. These rates suggested that survival of immature females was better than males. Beginning in 1989, survival of immature seals at French Frigate Shoals declined sharply.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.     

Exploiting the complete yeast genome sequence

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.     

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.