Human mitochondrial glutamic-oxaloacetic-transaminase,GOTM: Formal genetics

Summary The polymorphism of human GOT_M was investigated in red blood cells by means of routine starch gel electrophoresis. The formal model of two common alleles, GOT _M ¹ and GOT _M ² , at an autosomal locus GOT_M was confirmed by examination of 640 mother-child pairs. The frequency of GOT _M ¹ in this sample from southwestern Germany was calculated to be 0.981±0.003.     

Pre-Anthesis Reserve Utilization for Protein and Carbohydrate Synthesis in Grains of Wheat

We assessed the contribution of pre-anthesis reserve C to protein and carbohydrate deposition in grains of wheat (Triticum aestivum L.) using a new approach comprised of steady-state ¹³C/¹²C labeling and separation of the protein and carbohydrate fractions of mature grains. Experiments were performed with two spring wheat cultivars (Kadett and Star) grown with differential N fertilizer supply over 2 years. Pre-anthesis reserves contributed between 30% and 47% of the C in protein and 8% to 27% of the C in carbohydrates of grains. Partitioning of pre-anthesis C among the grain fractions was strongly dependent on the C/N (w/w) ratio in mobilized pre-anthesis biomass (r² = 0.92). There appeared to be no significant exchange of pre-anthesis C between amino acids and carbohydrates during redistribution. The mean apparent efficiency of mobilized carbohydrate-C use in grain filling (ME_CHO, estimated as the mass of pre-anthesis C deposited in grain carbohydrates per gram of pre-anthesis C mobilized from carbohydrates in vegetative plant parts) was 0.72, whereas that of protein-C (ME_P) was 0.56. However, ME_P and ME_CHO varied among treatments. ME_CHO increased with increasing contributions of water-soluble carbohydrates to total pre-anthesis carbohydrate mobilization. ME_P decreased with increasing residence time of protein in vegetative biomass. Possible causes for variability of ME_P and ME_CHO are discussed.     

Effect of dietary protein and energy intake on embryonic survival and gene expression in the uterine endometrium of early pregnant gilts

Porcine embryonic loss during early gestation is a serious problem in swine production. Improving embryonic survival can be achieved by maternal manipulation. Protein and energy are two major components of the diet, which play decisive roles in embryonic survival. This study was performed to evaluate the effects of enhancing maternal protein or energy intake on embryonic survival during early gestation in gilts and to explore the underlying mechanism. From day (d) 0 to 30 of gestation, 40 gilts (Landrace × York) were randomly allocated to 5 diets according to daily intake of low (L, National Research Council (NRC) recommendation for gestation gilts), medium (M, 20% higher than NRC) or high (H, 40% higher than NRC) CP or metabolisable energy (ME) (L_CPL_ME, M_CPL_ME, H_CPL_ME, L_CPH_ME, H_CPH_ME). Gilts were sacrificed on d 30 of gestation, and number of foetuses and corpora lutea, embryonic survival rate, uterine weight, and total volume of allantoic fluid were recorded or calculated. Gene expression was determined by Quantitative Real-time PCR (qPCR), western blot or immunohistochemistry. Results showed that increasing protein or ME intake significantly increased embryonic survival rate. Compared with diet L_CPL_ME, plasma progesterone (P4) concentration in diet L_CPH_ME increased at d 14 and d 30 of gestation. Progesterone receptor (PGR) was found not to be expressed in the epithelia but was strongly expressed in the stroma of the endometrium. Increasing protein or ME intake did not alter PGR expression in the endometrium. There was also no change in the amount of P4, hepatocyte growth factor, and fibroblast growth factor-7 in the endometrium. The mRNA abundance of cationic amino acid transporter 1 in the endometrium in diet L_CPH_ME and H_CPH_ME was significantly lower than in diet L_CPL_ME. Diet H_CPL_ME showed a tendency to increase neutral amino acid transporter 1 mRNA expression in the endometrium compared to diet L_CPL_ME (P = 0.087). In conclusion, increasing maternal protein or ME intake had a positive effect on the embryonic survival. Increased protein intake by 20 or 40% did not alter plasma P4 level, but increasing ME intake by 40% improved plasma P4 concentration at d 14 and 30 of gestation. Increasing maternal protein or ME intake did not induce PGR expression in the endometrium. Maternal protein and energy intake likely mediate transportation of cationic and neutral amino acids from mother to foetus to affect embryonic survival and development.     

The positively cooperative binding of concanavalin a to corn starch: The isotherm of binding and a measure of the cooperativity

The binding of concanavalin A to corn starch was investigated by fluorimetric assay. The extent of binding varied linearly with the mass of ligand, and followed a hyperbolic law with respect to the mass of starch. This led to an isotherm of binding: r = 0.33A_oME_o^?0.88, where r is the extent of binding, A_o is the mass of concanavalin A present (both bound and unbound), and M_o is the mass of starch. These results, and Scatchard plots of the data, showed the binding to be positively cooperative. The exponent of the M_o term was shown to be a measure of cooperativity. The binding was dependent on the ionic strength of the dispersion medium, and this indicated that the binding may have an electrostatic component.     

Contact inhibition--plasma membranes enzymatic activities in cultured cell lines

Two phenotypically stabilized lines of plasmocytoma MOPC 173 - one of which (ME₂) is contact inhibited; the other (MF₂) is not - have different patterns of plasma membrane enzymatic activities. Mg⁺⁺K⁺ Na⁺ dependent ATPase and 5′ nucleotidase activities of ME₂ decrease sharply when cells come into contact.     

松叶猪毛菜NADP-苹果酸酶基因家族及启动子克隆分析北大核心CSCD

NADP-苹果酸酶(NADP-ME)是C_(4)植物的关键光合酶,在生物和非生物胁迫中发挥了重要的作用。为了进一步研究该酶编码基因的功能,该研究以典型荒漠C_(3)-C_(4)中间型植物松叶猪毛菜为研究对象,在克隆得到NADP-ME家族基因序列的基础上,研究其表达部位及在非生物胁迫下的表达模式,并克隆其启动子序列分析响应非生物胁迫的元件差异。结果表明:(1)成功获得松叶猪毛菜3个NADP-MEs,命名为SaNADP-ME1、SaNADP-ME2和SaNADP-ME4,CDS序列长度分别为1755、1758和1941 bp。(2)SaNADP-ME1主要在根中表达,SaNADP-ME2和SaNADP-ME4主要在叶中表达;在ABA、NaCl、NAHCO_(3)和PEG_(6000)胁迫下松叶猪毛菜幼苗中3个NADP-MEs均可被诱导表达,且SaNADP-ME2和SaNADP-ME4的响应表达模式相似。(3)成功克隆得到SaNADP-ME1、SaNADP-ME2和SaNADP-ME4启动子区域2351、1655和2887 bp。生物信息学分析发现它们都含有基本启动子元件以及响应外界刺激的元件。     

Determination of maintenance energy requirement and responses of dry ewes to dietary inclusion of lucerne versus concentrate meal

An accurate value for metabolizable energy (ME) requirement for maintenance (ME_m) is essential to enable sheep husbandry practice to reach its potential. The objectives of the study were to use calorimetry chamber data of dry ewes (Hu × thin-tail Han F1 crossbred) to develop updated ME_m, examine effects of substituting concentrate feed with lucerne hay on energy partitioning, and explore the relationships between energy utilization and fasting heat production (FHP). Data were collected from three experiments. In Exps. 1, 2a and 2b, lucerne hay was used to replace concentrates in three levels (0:40%, 15:25% and 30:10%), with diets containing 60% maize stover (Exp. 1), fresh rye forage (Exp. 2a) or dry rye forage (Exp. 2b). Within each experiment, diets were isoenergetic (digestible energy, DE) and isonitrogenous. Exp. 3 aimed at evaluating effects of three BW levels on nutrient utilization of dry ewes offered diets containing 60% maize stover, 15% lucerne hay and 25% concentrates. Energy metabolism data were measured using the respiration calorimeter chamber technique in all three experiments, followed by the measurement of FHP in Exps. 1, 2b and 3. The ME_m derived from the linear regression between energy balance (EB) and ME intake was 0.440 MJ/kg BW^0.75. The average FHP was 0.326 MJ/kg BW^0.75. The fasting metabolism, net energy requirement for maintenance (NE_m) and ME_m were estimated to be 0.336, 0.359 and 0.511 MJ/kg BW^0.75, respectively, through adjustment of FHP using fasting urinary energy output, activity allowance and efficiency of ME use for maintenance. The FHP was negatively correlated to EB/metabolic BW, ME/gross energy (GE), ME/DE, EB/GE intake and EB/ME intake, while positively correlated to HP/GE intake, HP/ME intake and CH₄-E/GE intake. Compared to zero lucerne hay diet, the 15% lucerne hay intake decreased HP (MJ/d), and had no negative effects on EB (MJ/d) or energy utilization efficiencies. The results indicate that nutrient requirement standards currently used across the world are likely to underestimate ME_m for dry ewes, and the selection of low FHP ewes for breeding has the potential to improve sheep production efficiency.     

Radioiodinated ligands for the estrogen receptor: Tissue distribution of 17α-[125I]iodovinylestradiol derivatives in normal and tumor-bearing adult female rats

A series of three ¹²⁵I-labeled 17α-iodovinylestradiol derivatives previously demonstrated to have a selectivity for estrogen receptor tissues in immature female rats were selected for further evaluation in adult female rats. In normal adult female rats, the iodovinyl analogs of moxestrol (IVβME₂) and moxestrol-3-O methyl ether (IVβME₂-3-OMe) demonstrated high uterine uptake (0.296–0.437 and 0.135–0.199%ID-kg/g) and selectivity (10–18:1) over the 6 h time period. Subsequent evaluation in two tumor models indicated that [¹²⁵I]VβME₂ also possessed the highest tumor uptake and selectivity in the adult female rats bearing the estrogen responsive 9,10-dimethyl-1,2-benzanthracene(DMBA)-induced mammary tumors and that this was receptor mediated. An estrogen independent tumor, the transplanted Walker 256 mammary adenocarcinoma, showed no selectivity of radioligand uptake compared with nontarget tissues. The results suggest the applicability of this agent to the in vivo detection and characterization of estrogen responsive tumors in man.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells

The K⁺ content and the K⁺ flux were measured in the cell lines ME₂ and MF₂ isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the same K⁺ content and the same K⁺ steady state flux per unit of surface area.In ME₂ cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K⁺ net movement.The (Na⁺ + K⁺)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K⁺ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells.The K⁺ net flux is considerably increased when the intracellular K⁺ level is reduced after preincubation of the cells in a K⁺-free medium. Thus, internal K⁺ seems to regulate the K⁺ influx.     

Immunogenic and Protective Properties of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> IgA1 Protease and of Its Truncated Fragments

Four recombinant proteins, MA²⁸–P¹⁰⁰⁴LEH₆, ME¹³⁵–H³²⁸LEH₆, MW³²⁹–H⁶²²LEH₆ and MH⁸³⁵–P¹⁰⁰⁴LEH₆, were prepared based on the genomic sequence of IgA1 protease from Neisseria meningitidis serogroup B strain H44/76. The immunogenic and protective properties of these proteins were studied in a mouse model. The predicted T- and B-epitopes located in the N-terminal part of amino acid sequence of this enzyme are very important for the formation of effective protection against meningococci of the three main epidemic serogroups A, B, and C. The small-sized recombinant protein having the sequence ME¹³⁵–H³²⁸LEH₆ (molecular weight 23367 Da) appears to be as protective against meningococci of the tested serogroups as the high molecular MA²⁸–P¹⁰⁰⁴LEH₆ (molecular weight 109019 Da), the latter being a large-sized analog of full-length IgA1 protease. These proteins can be promising candidates for a polyvalent meningococcal vaccine.     

Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me₂SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME₂SO, 1M ME₂SO + 1M EG, and 1M ME₂SO + 0.5M EG). Next, fresh islets, 2M ME₂SO islets, and 1M ME₂SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME₂SO (1M ME₂SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME₂SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME₂SO + 0.5M EG), compared to islets preserved with 2M ME₂SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.     

Gene controlled selection of mitochondria in Paramecium.

Summary The slow growing mutant cl₁ of Paramecium, previously described (Sainsard, Claisse and Balmefrezol, 1974) differs from wild-type by a single recessive nuclear mutation and by a particular mitochondrial phenotype (M^cl) that gene cl ₁ distinguishes from the wild-type mitochondrial phenotype (M⁺). A further analysis of these nucleo-mitochondrial interactions was carried out by confronting the genes cl ₁ and cl ₁ ⁺ with mixed populations of M⁺ and M^cl mitochondria obtained after cytoplasmic exchange at conjugation. The following results were obtained: 1. M⁺ and M^cl mitochondria introduced respectively into mutant and wild-type cells do not multiply easily; 2. when a mixed population (M⁺+M^cl) is established, both mitochondrial types are maintained during the growth of the F₁ heterozygous cl ₁/cl ₁ ⁺ clones; 3. when the nuclear segregation occurs in F₂, the formation of homozygotes cl ₁/cl ₁ or cl ₁ ⁺ /cl ₁ ⁺ is soon followed by the segregation of the two mitochondrial types, M^cl or M⁺, reconstituting the two parental nucleo-mitochondrial associations.This paper is dedicated to Professor T.M. Sonneborn on the occasion of his 70th birthday     

Binding of imidazole and 2-methylimidazole by hemes in organic solvents. Evidence for five-coordination

By means of absorption spectroscopy we show that in benzene solutions, only one molecule of 2-methylimidazole is bound with a great affinity by deuteroheme (K = 1.25 10⁴ M^?1) and mesotetraphenylheme (K = 2.4 10⁴ M^?1). Besides, two overlapping steps may be distinguished when hemes bind imidazole molecules. The equilibrium constants are K₁ = 4.5 10³ M^?1 and 8.8 10³ M^?1, K₂ = 6.8 10⁴ M^?1 and 7.9 10⁴ M^?1 for deuteroheme and mesotetraphenylheme respectively.     

Interlocked DNA rings. II. Physicochemical studies

Several species of topologically interlocked λb2b5c DNA rings (catenanes) have been prepared in vitro. The sedimentation behavior of dimeric catenanes containing 0, 1, or 2 covalently closed duplex rings has been studied as a function of the superhelical density of the covalently closed ring or rings. In general, if XtpY represents rings X and Y topologically interlocked, the sedimentation coefficient of this species, ^SXtpY, is related to the sedimentation coefficients ^SX and ^SY of the component rings by the empirical relationship ^SXtpY/^SY = [(M_X + M_Y)/M_Y] [1 + (M_X/M_Y)^1.78(^SY/^SX)^1.78]^?0.56 This equation can also be extended to the case where three monomeric rings are topologically linked in a chain. For two topologically interlocked monomeric rings with neither ring covalently closed, the sedimentation coefficient is 1.35 times that of the monomeric ring. This is different from results reported for mitochondrial and P22 catenanes by others. Several possibilities are discussed to account for this discrepancy. The sedimentation coefficient of a species containing one covalently closed duplex ring and a single-stranded ring was also measured in an alkaline medium. This species, which can be easily derived from a dimeric catenane containing two covalently closed duplex rings, is particularly useful for the identification of covalently closed dimeric catenanes. Some electron microscopic studies of these interlocked rings are presented in an accompanying paper.     

Molecular characterization of a heat-modifiable protein from the outer membrane of Escherichia coli.

Discrete fractions of nonhistone chromosomal proteins (NHCP) were obtained from rabbit liver chromatin by their dissociation in 5 m urea with increasing concentrations of NaCl. Three fractions were obtained: M₀, M₁, and M₃. We found that M₀ can modify the conformation of DNA/histone complexes as depicted from their induced increase in the ellipticity of DNA/histone from 5100 to 6900 degree-cm²/dmol. This effect was found to be reversible, while M₁ and M₃ effects, if any, were not measurable. These results suggest that M₀ primarily interacts with the chromatin subunit.     

Normolipidemic Effect of Antioxidants in Low Cholesterol-Modified Poultry Egg<Superscript>Ψ</Superscript> on Zn-induced Dyslipidemia and Liver Pathology in Wistar Rats

A low cholesterol (CH)-modified poultry egg (ME^Ψ) containing more vitamin E, lenolenic acid, and minerals Cu and Mg but low total lipid (TL) and Zn contents than the conventional egg evaluated to reduce the severity of dyslipidemia induced by excessive Zn in the diet. The experimental data was recorded on male rats fed on normolipidemic (NL) semi-synthetic basal diet containing 20 mg Zn/kg diet in control group I, Zn supplemented dyslipidemic diet-A (Zn-DL-A) and B (Zn-DL-B) containing 40 and 80 mg Zn/kg diet in groups II and III, and ME^Ψ-mixed Test diet-A (Zn-DL-A + 4 ME^Ψ) and Test diet-B (Zn-DL-B + 4 ME^Ψ) in groups IIEM and IIIEM, respectively, for 180 and 90 days. Data recorded on liver and blood lipid profiles showed reduction in the concentration of TL, CH, triglycerides, and glycogen (GG) in liver consequently leading to their rise in blood serum including rise in VLDL-c and LDL-c but fall in HDL-c in groups II and III rats that reversed after ME^Ψ treatment resulting in rise of their levels in the liver and fall in the blood of groups IIEM and IIIEM rats, respectively. Mineral status in the liver showed a rise in Zn but fall in Cu and Mg levels in groups II and III that was reversed after ME^Ψ treatment resulting in fall in Zn and rise in Cu and Mg concentration in the liver of groups IIEM and IIIEM rats. Hepatopathogical studies showed reduction in the dilatation of long citernal profile of endoplasmic reticulum and increase in GG and TL granules in the cytoplasm of hepatocytes of groups IIEM and IIIEM after ME^Ψ treatment than those of groups II and III rats. It was concluded that the inclusion of ME^Ψ would be helpful in reducing dyslipidemia by correcting the ionic imbalance generated by excessive Zn intake in rats or by drugs, even in chronic diseased conditions without aggravating risk factors for heart diseases in humans that need further studies.     

Equilibrium studies on neutral red-DNA binding.

Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The pK_A of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 10³ M^?1 and 0.136 × 10³ M^?1, respectively, for native DNA and 7.48 × 10³ M^?1 and 0.938 × 10³ M^?1, respectively, for denatured DNA. The pK_A value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.     

Alginate beads as a tool to handle,cryopreserve and culture isolated human primordial/primary follicles

Background

One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME₂SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate.

Methods

Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5–15 per group). Follicles were frozen-thawed using 1.4 M ME₂SO or 1.5 M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte.

Results

A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME₂SO (n = 424) or EG (n = 259), or used as controls (n = 158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME₂SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME₂SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME₂SO groups.

Conclusions

Our results show that 1.4 M ME₂SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5 M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME₂SO as a CPA.