Urinary excretion of isomers of biliverdin after destruction in vivo of haemoproteins and haemin.

The amount and isomeric composition of urinary biliverdin in rabbits were analysed by h.p.l.c. Physiological values were maintained after the injection of haemin. On the other hand, when haemoglobins from several mammalian species were injected into rabbits, the excretion of biliverdin-IX alpha and biliverdin-IX beta were increased 6-18-fold and 32-66-fold respectively over physiological excretion. Injection of myoglobin resulted in a 44-fold increase in excretion of the IX alpha-isomer. Coupled oxidation with ascorbate of haemoglobin and myoglobin by oxygen produced mainly the IX alpha- and IX beta-isomers from haemoglobin and the IX alpha-isomer from myoglobin. The destruction of part of the haem from injected haemoproteins by non-enzymic chemical degradation would account for the observed respective increases in the excretion of biliverdin isomers. The excretion of biliverdin isomers after the injection of phenylhydrazine into rabbits was similar to that after the injection of haemoglobin.     

Consequences of haemolysis without haptoglobin

Haptoglobin (Hp), a conserved plasma glycoprotein, forms very stable soluble complexes with free plasma haemoglobin. Haemoglobin binding by haptoglobin is thought to be important in the rapid hepatic clearance of haemoglobin from the plasma and in the inhibition of glomerular filtration of haemoglobin. It is thought to reduce haemoglobin-induced renal damage during haemolysis. To evaluate these functions, Hp knockout (Hp-/-) mice were created. The Hp-/- mouse was generated by a standard gene replacement technique in mouse embryonic stem cells. These mice were evaluated with and without haemolysis using several parameters: mortality, haemoglobin clearance, renal tissue damage and function. Hp-/- mice were viable but had a small, significant reduction in postnatal viability. The lack of Hp did not impair clearance of free plasma haemoglobin. Induction of severe haemolysis by phenylhydrazine caused extensive haemoglobin precipitation in the renal tubular cells. However, haemoglobin precipitation in the kidney was not increased in Hp-/- mice. Nevertheless, Hp-/- mice were more susceptible to phenylhydrazine with a mortality rate of 55% in Hp-/- mice versus 18% in Hp+/+ mice. In general, phenylhydrazine-treated Hp-/- mice suffered greater tissue damage, as evidenced by the induction of a hepatic acute phase response, resulting in increased plasma alpha1-acidic glycoprotein (AGP) levels and higher plasma malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (HNE) levels. Gross pathological analysis indicated that the kidney was the most affected tissue in phenylhydrazine-treated Hp-/- and Hp+/+ mice, and Hp-/- mice were more severely affected. They had lower mitotic indices in their kidneys, higher basal levels of renal lipid peroxidation, as evidenced by levels of malonaldehyde and 4-hydroxy-2(E)-nonenal (MDA/HNE) and elevated levels of 8-hydroxyguanine (but not other products of oxidative DNA damage). There also was increased induction of haem oxygenase-1. The more severe renal damage in Hp-/- mice was also evident in the delayed erythropoietin gene expression and poorer renal clearance of [3H]-inulin. The reduction in glomerular filtration function in Hp+/+ and Hp-/- mice could be restored to baseline by vasodilators (prazosin or diazoxide), implicating renal vasoconstriction as a major mechanism of acute renal failure during induced haemolysis. These data suggest that Hp plays a pivotal role in reducing renal oxidative damage during haemolysis.     

Electrochemical investigation of the effect of some organic phosphates on haemoglobin

The effects of DPG, IHP, GTP, GDP and GMP on the structure and stability of haemoglobin were electrochemically investigated with an iodide-modified silver electrode in 0.01 M KNO₃ at pH 7.0. Anodic and cathodic peaks of haemoglobin were observed at 250 mV and 12 mV with a formal potential value of 133 mV vs. Ag/AgCl. The effects of different concentrations of DPG, IHP, GTP, GDP and GMP on the anaerobic redox reaction were determined. The results showed that DPG and IHP can lead to a positive shift in the reduction peak of haemoglobin, indicating that the oxidation peak shift of haemoglobin was small as a result of stabilization of the reduced state and destabilization of the R-like state of haemoglobin. GTP elicited a more positive shift in the cathodic and anodic peaks of haemoglobin at a higher concentration, signifying that it has a low-affinity binding site on haemoglobin. The positive shift of the cathodic and anodic peaks revealed a slight variation in the structure and indicated the unfolding of haemoglobin in the presence of high concentrations of GTP. Our study also showed that GDP and GMP did not cause significant shift the cathodic and anodic peaks of haemoglobin even at high concentrations, refuting the existence of specific GDP-and GMP-binding sites on the protein. Moreover, the iodide-modified silver electrode method proved to be easy and useful in investigating the effects of ligands or other effectors on haemoglobin in solution.     

Haemoglobin denaturation, lipid peroxidation and haemolysis in phenylhydrazine-induced anaemia

Temporal changes in the levels of denatured haemoglobin (Heinz bodies) and fluorescent lipid peroxidation products in the red cells of rabbits administered phenylhydrazine have been followed. Heinz bodies were maximal just before the period when most of the cell destruction occurred, whereas lipid peroxidation products were maximum when reticulocyte levels were highest. This implies that lipid peroxidation occurs mainly in immature cells and that haemoglobin denaturation is more likely than lipid peroxidation to be a major contributor to haemolysis.     

Consequences of haemolysis without haptoglobin

Abstract

Haptoglobin (Hp), a conserved plasma glycoprotein, forms very stable soluble complexes with free plasma haemoglobin. Haemoglobin binding by haptoglobin is thought to be important in the rapid hepatic clearance of haemoglobin from the plasma and in the inhibition of glomerular filtration of haemoglobin. It is thought to reduce haemoglobin-induced renal damage during haemolysis. To evaluate these functions, Hp knockout (Hp^-/-) mice were created. The Hp^-/- mouse was generated by a standard gene replacement technique in mouse embryonic stem cells. These mice were evaluated with and without haemolysis using several parameters: mortality, haemoglobin clearance, renal tissue damage and function.

Hp^-/- mice were viable but had a small, significant reduction in postnatal viability. The lack of Hp did not impair clearance of free plasma haemoglobin. Induction of severe haemolysis by phenylhydrazine caused extensive haemoglobin precipitation in the renal tubular cells. However, haemoglobin precipitation in the kidney was not increased in Hp^-/- mice. Nevertheless, Hp^-/- mice were more susceptible to phenylhydrazine with a mortality rate of 55% in Hp^-/- mice versus 18% in Hp^+/+ mice. In general, phenylhydrazine-treated Hp^-/- mice suffered greater tissue damage, as evidenced by the induction of a hepatic acute phase response, resulting in increased plasma₁-acidic glycoprotein (AGP) levels and higher plasma malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (HNE) levels. Gross pathological analysis indicated that the kidney was the most affected tissue in phenylhydrazine-treated Hp^-/- and Hp^+/+ mice, and Hp^-/- mice were more severely affected. They had lower mitotic indices in their kidneys, higher basal levels of renal lipid peroxidation, as evidenced by levels of malonaldehyde and 4-hydroxy-2(E)-nonenal (MDA/HNE) and elevated levels of 8-hydroxyguanine (but not other products of oxidative DNA damage). There also was increased induction of haem oxygenase-1. The more severe renal damage in Hp^-/- mice was also evident in the delayed erythropoietin gene expression and poorer renal clearance of [³H]-inulin. The reduction in glomerular filtration function in Hp ^+/+ and Hp^-/- mice could be restored to baseline by vasodilators (prazosin or diazoxide), implicating renal vasoconstriction as a major mechanism of acute renal failure during induced haemolysis.

These data suggest that Hp plays a pivotal role in reducing renal oxidative damage during haemolysis.     

Effect of high fluoride intake on haematological aspects of the mouse.

The effect of feeding adult Swiss albino mice of both sexes a diet supplemented with 0, 125, 250 and 500 parts/10(6) of fluoride for four and eight week periods on haemoglobin concentration (Hb), packed cell volume (PCV) and mean corpuscular haemoglobin concentration (MCHC) was investigated. Values of the three parameters were significantly lowered at both periods in the treated groups as compared with the controls. The extent of reduction in these values was, in general, dependent on the dose of supplemented dietary fluoride. Clinical symptoms were not observed before the end of the sixth week. However, appearance of the symptoms did not change the trend of variations in Hb, PCV and MCHC values. The reduced values could be the result of lowered haemoglobin synthesis and erythropoiesis. It was suggested that these haematological indices could serve to detect preclinical effects of high fluoride intake with an added dose of as low as 125 parts/10(6), or even less, for a period of four weeks or probably earlier.     

表达重组猪瘟病毒E290多肽的干酪乳杆菌口服免疫特性及诱导特异性CTL反应的研究

经PCR扩增获得约60bp编码猪瘟病毒T细胞表位E290多肽基因片段,克隆至表达载体pPG-VP2中VP2基因5′端上游,命名为pPG-VP2-E290,电转化干酪乳杆菌,构建了表达猪瘟病毒E290多肽的重组乳酸菌系统。口服免疫BALB/c鼠和新西兰兔,检测诱导小鼠和兔体内产生特异性抗猪瘟病毒E290多肽IgG水平,并对E290多肽的CTL活性进行检测,同时对免疫兔进行猪瘟病毒攻毒实验,检测E290多肽抗体对免疫兔的保护作用。构建的重组猪瘟病毒T细胞表位的干酪乳杆菌具有良好的免疫性,口服免疫后的小鼠和兔血清中均检测到了较高水平的抗E290多肽抗体IgG,且能诱导小鼠机体产生抗猪瘟病毒的特异性CTL反应,亦证实猪瘟病毒E290免疫兔能够抵抗猪瘟病毒的攻击。     

Haemoglobin variants in mice.

Haemoglobin variants were studied in wild and laboratory house mice (Mus musculus), including standard and new inbred strains, using starch-gel electrophoretic technique. Single (Hbbs) or diffuse (Hbbd) types of haemoglobin were found in all of them. The embryonic haemoglobin pattern was different from although similar to that of the adult in all the strains. The haemoglobins revealed monomorphism in the inbred strains, while polymorphism was observed in non-inbred laboratory and wild mice.     

几种免疫增强剂对睡眠的影响及其机制

本文研究了几种免疫增强剂对动物睡眠的影响,并分析了免疫系统影响睡眠的物质基础。异丙肌苷、转移因子、胞壁酰二肽（ＭＤＰ）在增强动物免疫功能的同时,均能延长动物的慢波睡眠时间。以ＭＤＰ为代表观察了肿瘤坏死因子（ＴＮＦ）在免疫系统影响睡眠过程中的中介作用。结果表明,ＴＮＦ可促进家兔睡眠;ＭＤＰ可通过促进星形胶持细胞中ＴＮＦ－αｍＲＮＡ的表达,增加ＴＮＦ的合成与释放,从而提高脑内ＴＮＦ水平;ＴＮＦ单克隆抗     

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

Anti-sickling, analgesic and anti-inflammatory properties of 3,5-dimethoxy-4-hydroxy benzoic acid and 2,3,4-trihydroxyacetophenone.

Effects of 3,5-dimethoxy-4-hydroxybenzoic acid and 2,3,4-trihydroxyacetophenone were studied on haemoglobin S (Hb S) polymerisation, analgesia and inflammation using Hb S solution, rats and mice. UV spectrophotometric procedure was used to monitor the polymerization of the Hb S. Acetic acid induced writhing in mice and egg albumin induced rat paw edema procedures were used to evaluate analgesic and anti-inflammatory activities of the compounds respectively. The results indicate that both drugs inhibit the process of polymerization significantly, possibly by direct action on the Hb S molecules. The drugs inhibited acetic acid induced pain and decreased egg albumin induced oedema. It is concluded that 3,5-dimethoxy-4-hydroxybenzoic acid and 2,3,4-trihydroxyacetophenone may have some value in the management of sickle cell disease.     

Comparison of carbonic anhydrase activity and other haematological values of two Eutherian and three Australian marsupial mammals

1. Carbonic anhydrase activity and 2,3-diphosphoglycerate (2,3-DPG) concentration were determined in whole blood from humans (Homo sapiens), rabbits (Oryctolagus cuniculus), eastern grey kangaroos (Macropus giganteus), pademelons (Thylogale billardierii) and brush-tailed possums (Trichosurus vulpecula). 2. Marsupial blood carbonic anhydrase activity increased as species body size decreased. 3. T. billardierii haemoglobin was found to have a polymorphism which may be the same (beta 2 = histidine or glutamine) as that of M. giganteus. 4. The concentration of 2,3-DPG int e red cells of T. billardierii was approximately equal to that of the haemoglobin tetramer. Levels of 2,3-DPG in the other species were similar to those previously reported.     

Studies on Anoplotaenia dasyuri Beddard, 1911 (Cestoda: Taeniidae), a parasite of the Tasmanian devil: Observations on the egg and metacestode

The ultrastructure and hatching mechanisms of the egg of Anoplotaenia dasyuri showed minor differences from those of other taeniid species. The embryophore disintegrated under the action of pepsin, but activity of the embryo did not occur until placed in an alkaline solution containing pancreatin. The addition of bile from various host species did not affect the hatching of eggs of A. Daysuri although it did increase the rate of hatching of eggs of Taenia pisiformis. Metacestodes were recovered from the hearts of experimentally infected possums (Trichosurus vulpeculd), kangaroos (Macropus giganteus and M.fuliginosus) and from the livers of mice and guinea pigs fed eggs of A. Daysuri. A wombat (Vombatus ursinus), rats and rabbits could not be infected orally. Hatched and activated embryos, injected intravenously, developed in the hearts of kangaroos and wallabies (M. rufogriseus), the livers, lungs, kidneys and hearts of mice, but not at all in rats and rabbits. Similarly, injections of embryos into the portal veins of mice, rats and rabbits resulted in development of metacestodes in the lungs and liver of mice only. The development of some stages of the metacestode are briefly described and the possible phylogeny of the parasite discussed.     

Studies on adjuvants for human prophylactics. III. Comparison of adjuvanticity in different animal species.

Activities of common adjuvants were compared in mice, rabbits and monkeys with tetanus toxoid as an antigen. Aluminium gel showed consistently high adjuvanticity for antitoxin production in all animal species examined when administered subcutaneously. Water-in-oil in water (w/o/w) showed high activity comparable to that of aluminium in mice and rabbits but no activity in monkeys. Endotoxin was considerably effective in rabbits and monkeys but not so in mice. Production of both IgM and IgG antitoxin was promoted by the effective adjuvants in rabbits and monkeys. In mice, however, the effects of adjuvants on the production of IgM antitoxin was less significant and inconsistent. Contrary to the case of guinea pigs, tetanus antitoxin was produced in mice by ip injection to a level comparable to that induced by sc injection. The effects of adjuvants in mice administered by ip and sc injection were not significantly different from each other.     

New insights into bioprotective effectiveness of disaccharides: an FTIR study of human haemoglobin aqueous solutions exposed to static magnetic fields

The aim of this study was the investigation of static magnetic field effects on haemoglobin secondary structure and the bioprotective effectiveness of two disaccharides, sucrose and trehalose. Samples of haemoglobin aqueous solutions, in the absence and in the presence of sucrose and trehalose, were exposed to a uniform magnetic field at 200 mT, which is the exposure limit established by the ICNIRP recommendation for occupational exposure. Spectral analysis by FTIR spectroscopy after 3 and 7 h of exposure revealed a decrease in the amide A vibration band for haemoglobin in bi-distilled water solution. Analogue exposures did not produce any appreciable change of amide A for haemoglobin in sucrose and trehalose solutions. Otherwise, no relative increase of \upbeta \upbeta -sheet contents in amide I and II regions was detected for haemoglobin aqueous solutions, leading us to exclude the hypothesis that static magnetic fields can induce the formation of aggregates in the protein. In addition, a decrease in CH₃ stretching linkages occurred for haemoglobin in bi-distilled water solution after exposure, which was not observed for haemoglobin in sucrose and trehalose aqueous solutions, providing further evidence of a bioprotective compensatory mechanism of such disaccharides.     

神经纤毛蛋白-1的原核表达及兔抗小鼠神经纤毛蛋白-1抗体免疫学活性的研究

目的:在原核系统中分段表达神经纤毛蛋白-1(Nrp1);将纯化的蛋白免疫家兔后获得特异的抗体,并将其应用于检测组织和细胞中Nrp1分子的表达。方法:提取BALB/c胎鼠脑组织总RNA,通过RT-PCR分段扩增获得Nrp1基因片段,将PCR产物插入表达载体pET28a( ),获得含5个Nrp1基因片段的重组质粒,在大肠杆菌BL21(DE3)中诱导蛋白表达并纯化;将纯化的重组蛋白免疫新西兰大白兔获得针对目的蛋白的特异性多克隆抗体;利用Nrp1特异性多抗检测胎鼠脑组织和HeLa细胞中Nrp1的表达。结果:在原核系统中分段表达了Nrp1蛋白,通过Ni-NTA纯化了Nrp1蛋白片段;纯化的Nrp1蛋白免疫新西兰大白兔获得了具有免疫活性的多抗;兔抗小鼠Nrp1特异性多抗可用于检测组织、真核细胞中Nrp1的表达。结论:应用原核系统成功地表达了Nrp1蛋白,兔抗小鼠Nrp1特异性多抗可用于免疫学检测Nrp1分子的表达。     

Immunocytochemical studies on parafollicular cells of various mammals

Using specific antisera, calcitonin, calcitonin gene-related peptide (CGRP), somatostatin as well as neuron-specific enolase, chromogranin, secretory peptide I and calbindin (vitamin D-dependent calcium-binding protein) were looked for in parafollicular cells of rats, Syrian hamsters, Mongolian gerbils, mice, guinea pigs, rabbits and pigs. Calcitonin and CGRP were most invariably present in various species. Somatostatin was absent in mice and Mongolian gerbils and present in variable amounts in the remaining species. Neuron-specific enolase could not be detected in rabbits, while in the pigs and the Mongolian gerbils it could be demonstrated only in some parafollicular cells. Calbindin was present exclusively in parafollicular cells of guinea pigs. Chromogranin and secretory protein-I were present only in some animal species.     

Evaluation of room cleaning procedures in a laboratory animal facility

Animal room cleaning procedures were developed that could be used routinely and economically in this animal facility. Bacterial samples from the floors of rooms housing rabbits, rats and mice provided a useful way to evaluate the effectiveness of the cleaning procedures, and to determine the in-use effectiveness of disinfectant solutions.     

Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method

A high efficient and simple transgenic technology on mice and rabbits to transfect spermatozoa with exogenous DNA/DMSO complex to obtain transgenic offspring, which is namely called DMSO-sperm mediated gene transfer (SMGT). Mouse sperm could be either directly transfected via injection into testis or cultured in vitro with the plasmed DNA containing the enhanced green fluorescent protein (EGFP) that could be expressed in the embryos and offspring. Then, 36 living transgenic rabbits were produced using the same technology, and the transgenic ratio of 56.3% was detected using PCR and Southern blot. As the controls, the transgenic ratios of 39.6% and 47.8% have also been tested using the liposomes mediated technology of Tfx-50 Reagent or Lipefectamin-2000, respectively. The results show that the female transgenic rabbits, as the mammary gland bioreactor models, could express the human tissue plasminogen activator mutant (htPAm) in their mammary cells when they are adult.