bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of transiently transfected reporter genes in 3T3 Swiss cells by the inducers of differentiation/apoptosis – dimethylsulfoxide, hexamethylene bisacetamide and trichostatin A
Authors: Ishiguro Kimiko , Sartorelli Alan C.
Published: June 2004

Despite decades of investigation, the primary site of action of the prototypical inducers of differentiation, dimethylsulfoxide and hexamethylene bisacetamide (HMBA), has not been delineated. During studies designed to analyze cis‐acting elements responsible for induction of stage‐specific globin genes, we discovered the capacity of HMBA and dimethylsulfoxide to enhance the expression of transiently transfected reporter genes derived from globin and nonglobin gene promoters, prominently in nonerythroid 3T3 Swiss cells. The action of HMBA and dimethylsulfoxide in the transient transfection system resembled that of the inhibitor of histone deacetylases (HDACs), trichostatin A (TSA), in that the three agents enhanced reporter gene expression (a) regardless of the promoter employed, (b) with similar kinetics and (c) with an increase in the steady‐state level of reporter mRNA. Transiently transfected DNA was assembled rapidly into a chromatinized structure in 3T3 cells, suggesting that transcription of reporter genes was at least in part repressed by chromatin organization. Nuclear run‐on analyses indicated that dimethylsulfoxide and HMBA enhanced transcriptional initiation of the reporter and p21/WAF1/Cip1 genes. In contrast, TSA produced negligible effects on nuclear run‐on transcription of these genes. HMBA and dimethylsulfoxide did not change the acetylation, phosphorylation, or methylation status of histones and did not activate stably transfected genes. Despite these differences, the three agents modulated the expression of common sets of cellular genes and induced differentiation or apoptosis in intact cells. The findings imply that HMBA and dimethylsulfoxide modulate transcription by a mechanism independent of histone acetylation.
2‐Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase–substrate interaction
Authors: Subach Oksana M. , Khoroshaev Anton V. , Gerasimov Dmitrii N. , Baskunov Vladimir B. , Shchyolkina Anna K. , Gromova Elizaveta S.
Published: June 2004

EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5′…CC*T/AGG…3′ DNA sequence and catalyzes the transfer of the methyl group from S‐adenosyl‐l‐methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2‐pyrimidinone, a cytosine analogue lacking an NH2 group at the C4 position of the pyrimidine ring. Also, DNA containing 2‐pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2‐Pyrimidinone was incorporated into the 5′…CCT/AGG…3′ sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2‐pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2‐pyrimidinone and to a greater destabilizing influence of 2‐pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2‐pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA–M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2‐Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S‐adenosyl‐l‐methionine, proton transfer to the C5 position of 2‐pyrimidinone occurs and in the presence of S‐adenosyl‐l‐methionine, methyl transfer to the C5 position of 2‐pyrimidinone occurs.
Evidence for two different electron transfer pathways in the same enzyme, nitrate reductase A from Escherichia coli
Authors: Giordani Roger , Buc Jean
Published: June 2004

In order to clarify the role of cytochrome in nitrate reductase we have performed spectrophotometric and stopped‐flow kinetic studies of reduction and oxidation of the cytochrome hemes with analogues of physiological quinones, using menadione as an analogue of menaquinone and duroquinone as an analogue of ubiquinone, and comparing the results with those obtained with dithionite. The spectrophotometric studies indicate that reduction of the cytochrome hemes varies according to the analogue of quinone used, and in no cases is it complete. Stopped‐flow kinetics of heme oxidation by potassium nitrate indicates that there are two distinct reactions, depending on whether the hemes were previously reduced by menadiol or by duroquinol. These results, and those of spectrophotometric studies of a mutant lacking the highest‐potential [Fe‐S] cluster, allow us to propose a two‐pathway electron transfer model for nitrate reductase A from Escherichia coli.
Protection of chylomicron remnants from oxidation by incorporation of probucol into the particles enhances their uptake by human macrophages and increases lipid accumulation in the cells
Authors: Moore Elizabeth H. , Napolitano Mariarosaria , Avella Michael , Bejta Fatos , Suckling Keith E. , Bravo Elena , Botham Kathleen M.
Published: June 2004

The effects of protection of chylomicron remnants from oxidation on their uptake and induction of lipid accumulation in macrophages were investigated using chylomicron remnant‐like particles (CRLPs) containing the lipophilic antioxidant drug, probucol, and macrophages derived from the human monocyte cell line, THP‐1. The total lipid content of THP‐1 macrophages was markedly higher (×2.2) after 48 h of incubation of THP‐1 macrophages with CRLPs containing probucol (pCRLPs) when compared to CRLPs without probucol, and this was because of increases in triacylglycerol (×2.3) and cholesterol (×1.8) levels, while cholesteryl ester concentrations were not significantly changed. Determination of the uptake of CRLPs and pCRLPs by the cells using particles labelled with the fluorescent probe 1,1′‐dioctadecyl‐3,3,3′3′‐tetramethylindo‐carbocyanine perchlorate showed that pCRLPs are taken up at a faster rate than CRLPs. The synthesis of triacylglycerol, as measured by the incorporation of [3H]oleate and [3H]glycerol, was also increased in macrophages incubated with pCRLPs as compared to CRLPs without probucol, but phospholipid and cholesteryl ester formation from [3H]oleate was unaffected. In addition, no differences between the effects of CRLPs and pCRLPs on the expression of mRNA for a range of genes believed to be involved in lipoprotein uptake, intracellular lipid metabolism and the efflux of cholesterol from macrophages was detected. These results suggest that antioxidants carried in chylomicron remnants enhance lipid accumulation in macrophages by increasing the rate of uptake of the particles and raising the intracellular synthesis of triacylglycerol, but not cholesteryl ester, and that these effects are brought about by changes at the post‐transcriptional level. Antioxidants carried in chylomicron remnants therefore may promote the development of atherosclerosis, and this is likely to be particularly important in conditions where clearance of remnants from the circulation is delayed.
Colocalization of insulin receptor and insulin receptor substrate‐1 to caveolae in primary human adipocytes
Authors: Karlsson Margareta , Thorn Hans , Danielsson Anna , Stenkula Karin G. , Öst Anita , Gustavsson Johanna , Nystrom Fredrik H. , Strålfors Peter
Published: June 2004

Caveolae are plasma membrane invaginations with several functions, one of which appears to be to organize receptor mediated signalling. Here we report that in primary human subcutaneous adipocytes the insulin receptor was localized to caveolae by electron microscopy/immunogold detection and by isolating caveolae from plasma membranes. Part of insulin receptor substrate 1 (IRS1), the immediate downstream signal mediator, was colocalized with the insulin receptor in the plasma membrane and caveolae, as demonstrated by immunofluorescence microscopy, immunogold electron microscopy, and immunogold electron microscopy of transfected recombinant HA‐IRS1. In contrast, rat epididymal adipocytes lacked IRS1 at the plasma membrane. Depletion of cholesterol from the cells using β‐cyclodextrin blocked insulin stimulation of glucose uptake, insulin inhibition of perilipin phosphorylation in response to isoproterenol, and insulin stimulation of protein kinase B and Map‐kinases extracellular signal‐related kinase (ERK)1/2 phosphorylation. Insulin‐stimulated phosphorylation of the insulin receptor and IRS1 was not affected, indicating that caveolae integrity is required downstream of IRS1. In conclusion we show that insulin receptor and IRS1 are both caveolar proteins and that caveolae are required for both metabolic and mitogenic control in human adipocytes. Our results establish caveolae as foci of insulin action and stress the importance of examining human cells in addition to animal cells and cell lines.
Temperature and concentration‐controlled dynamics of rhizobial small heat shock proteins
Authors: Lentze Nicolas , Aquilina J. Andrew , Lindbauer Mareike , Robinson Carol V. , Narberhaus Franz
Published: June 2004

A hallmark of α‐crystallin‐type small heat shock proteins (sHsps) is their highly dynamic oligomeric structure which promotes intermolecular interactions involved in subunit exchange and substrate binding (chaperone‐like activity). We studied the oligomeric features of two classes of bacterial sHsps by size exclusion chromatography and nanoelectrospray mass spectrometry. Proteins of both classes formed large complexes that rapidly dissociated upon dilution and at physiologically relevant heat shock temperatures. As the secondary structure was not perturbed, temperature‐ and concentration‐dependent dissociations were fully reversible. Complexes formed between sHsps and the model substrate citrate synthase were stable and exceeded the size of sHsp oligomers. Small Hsps, mutated in a highly conserved glycine residue at the C‐terminal end of the α‐crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild‐type proteins. Reduced complex stability coincided with reduced chaperone activity.
Human salivary α‐amylase Trp58 situated at subsite −2 is critical for enzyme activity
Authors: Ramasubbu Narayanan , Ragunath Chandran , Mishra Prasunkumar J. , Thomas Leonard M. , Gyémánt Gyöngyi , Kandra Lili
Published: June 2004

The nonreducing end of the substrate‐binding site of human salivary α‐amylase contains two residues Trp58 and Trp59, which belong to β2–α2 loop of the catalytic (β/α)8 barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild‐type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150–180‐fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in kcat, an increase in Km and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild‐type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP‐labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites −2, −3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites −2 and −3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose‐derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary α‐amylase.
The crystal structure of Thermoactinomyces vulgaris R‐47 α‐amylase II (TVA II) complexed with transglycosylated product
Authors: Mizuno Masahiro , Tonozuka Takashi , Uechi Akiko , Ohtaki Akashi , Ichikawa Kazuhiro , Kamitori Shigehiro , Nishikawa Atsushi , Sakano Yoshiyuki
Published: June 2004

Αn α‐amylase (TVA II) from Thermoactinomyces vulgaris R‐47 efficiently hydrolyzes α‐1,4‐glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes α‐1,4‐glucosidic linkages of cyclodextrins and α‐1,6‐glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 43‐α‐panosylpanose (43‐P2) (a pullulan hydrolysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 42‐α‐panosylpanose (42‐P2), which was produced by transglycosylation from 43‐P2, at 2.2‐Å resolution. The shape of the active cleft of TVA II is unique among those of α‐amylase family enzymes due to a loop (residues 193–218) that is located at the end of the cleft around the nonreducing region and forms a ‘dam’‐like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate‐liganded state and the other is supplying the water that is necessary for substrate hydrolysis.
The role of ADAM10 and ADAM17 in the ectodomain shedding of angiotensin converting enzyme and the amyloid precursor protein
Authors: Allinson Tobias M. J. , Parkin Edward T. , Condon Thomas P. , Schwager Sylva L. U. , Sturrock Edward D. , Turner Anthony J. , Hooper Nigel M.
Published: June 2004

Numerous transmembrane proteins, including the blood pressure regulating angiotensin converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein (APP), are proteolytically shed from the plasma membrane by metalloproteases. We have used an antisense oligonucleotide (ASO) approach to delineate the role of ADAM10 and tumour necrosis factor‐α converting enzyme (TACE; ADAM17) in the ectodomain shedding of ACE and APP from human SH‐SY5Y cells. Although the ADAM10 ASO and TACE ASO significantly reduced (> 81%) their respective mRNA levels and reduced the α‐secretase shedding of APP by 60% and 30%, respectively, neither ASO reduced the shedding of ACE. The mercurial compound 4‐aminophenylmercuric acetate (APMA) stimulated the shedding of ACE but not of APP. The APMA‐stimulated secretase cleaved ACE at the same Arg‐Ser bond in the juxtamembrane stalk as the constitutive secretase but was more sensitive to inhibition by a hydroxamate‐based compound. The APMA‐activated shedding of ACE was not reduced by the ADAM10 or TACE ASOs. These results indicate that neither ADAM10 nor TACE are involved in the shedding of ACE and that APMA, which activates a distinct ACE secretase, is the first pharmacological agent to distinguish between the shedding of ACE and APP.
Thermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395
Authors: Dunford Adrian J. , Marshall Ker R. , Munro Andrew W. , Scrutton Nigel S.
Published: June 2004

In rat neuronal nitric oxide synthase, Phe1395 is positioned over the FAD isoalloxazine ring. This is replaced by Trp676 in human cytochrome P450 reductase, a tryptophan in related diflavin reductases (e.g. methionine synthase reductase and novel reductase 1), and tyrosine in plant ferredoxin‐NADP+ reductase. Trp676 in human cytochrome P450 reductase is conformationally mobile, and plays a key role in enzyme reduction. Mutagenesis of Trp676 to alanine results in a functional NADH‐dependent reductase. Herein, we describe studies of rat neuronal nitric oxide synthase FAD domains, in which the aromatic shielding residue Phe1395 is replaced by tryptophan, alanine and serine. In steady‐state assays the F1395A and F1395S domains have a greater preference for NADH compared with F1395W and wild‐type. Stopped‐flow studies indicate flavin reduction by NADH is significantly faster with F1395S and F1395A domains, suggesting that this contributes to altered preference in coenzyme specificity. Unlike cytochrome P450 reductase, the switch in coenzyme specificity is not attributed to differential binding of NADPH and NADH, but probably results from improved geometry for hydride transfer in the F1395S– and F1395A–NADH complexes. Potentiometry indicates that the substitutions do not significantly perturb thermodynamic properties of the FAD, although considerable changes in electronic absorption properties are observed in oxidized F1395A and F1395S, consistent with changes in hydrophobicity of the flavin environment. In wild‐type and F1395W FAD domains, prolonged incubation with NADPH results in development of the neutral blue semiquinone FAD species. This reaction is suppressed in the mutant FAD domains lacking the shielding aromatic residue.
Mediator is required for activated transcription in a Schizosaccharomyces pombe in vitro system
Authors: Tamayo Evelyn , Bernal Giuliano , Teno Ursula , Maldonado Edio
Published: June 2004

RNA polymerase II (RNAPII) requires a set of general transcription factors − TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH − to initiate transcription from a gene promoter in vitro. General transcription factors have been isolated from Saccharomyces cerevisiae, rat, human and Drosophila, and their corresponding cDNAs have been cloned. In this report, we describe a reconstituted in vitro transcription system that consists of the following preparations of factors from the yeast Schizosaccharomyces pombe: affinity‐purified RNAPII, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF. We show that this system can support basal transcription from the adenovirus major late promoter when purified RNAPII is used and activated transcription when the RNAPII holoenzyme (RNAPII plus the Mediator proteins) is included in the reaction. In contrast, the TATA binding protein‐associated factors had no effect on transcriptional activation in our Sc. pombe system. These results indicate that Sc. pombe uses the same set of general transcription factors as other eukaryotes and that the Mediator is involved in activated transcription.
Physical resistance as a criterion in the selection of foraging material byAcromyrmex subterraneus brunneusForel, 1911 (Hym., Formicidae)
Authors: Camargo R. S. , Forti L. C. , de Matos C. A. O. , Lopes J. F. , de Andrade A. P. P.
Published: June 2004

Abstract: Leaf‐cutting ants forage on a wide variety of plant species using the physical and chemical characteristics of the plants as a selection criterion. In order to determine the influence of the physical factor on the selection of foraging material, inert materials such as floral sponge, polystyrene, plastic and clay, which possess different degrees of physical resistance to cutting, were offered simultaneously to fiveAcromyrmex subterraneus brunneuscolonies, and assessed 12 and 24 h after foraging. No substrate selectivity was observed during foraging. Physical resistance was used as a decision criterion for the incorporation or return of the foraged material. This fact suggests the existence of a second time of selection of the foraged material inside the colony during cultivation of the symbiontic fungus.
Relative toxicity of phosphine against the bean bruchid Acanthoscelides obtectus (Say) (Col., Bruchidae)
Authors: Hasan M. M. , Reichmuth C.
Published: June 2004

Abstract:  The toxicity of phosphine against adult Acanthoscelides obtectus (Say) was studied over a range of concentrations, viz. 0.01, 0.0125, 0.015, 0.02, 0.04, 0.08, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mg/l and exposure periods, viz. 0.25, 0.50, 1, 2, 6, 12, 24 and 48 h at 25°C and 65% relative humidity. Results show that both the parameters exhibited significant effects on the mortality of A. obtectus. Data indicate that 100% mortality only occurred in A. obtectus when 1‐, 2‐ and 12‐h exposure times were involved in the respective highest individual ct product. The ct products decreased as the exposure times increased for both the mortality levels of 50 and 99%. The minimum ct products for 50 and 99% kill were 0.19 and 0.29 mg h/l, respectively, at an exposure of 12 h. The influence of thresholds on regression equations of the form cnt = k were 0.59 and 13.80 (mg/l)nh for the fixed concentration for 50 and 99% mortality levels, respectively while they were 0.45 and 12.45 (mg/l)nh for the six exposure periods for the same mortality levels.
A probable cause of paradoxical thrombosis in zygomycosis
Authors: Rüchel R. , Elsner C. , Spreer A.
Published: June 2004

Invasive zygomycoses (syn. mucormycoses) are rather rare but life‐threatening diseases which often take a peracute course. Particularly endangered are diabetics and patients suffering from siderophilia. Zygomycosis is regularly complicated by thrombosis and subsequent necrosis. Usually it evolves from sinusitis in a rhinocerebral form. With the use of a clinical isolate (Rhizopus microsporus) and sera of the same female survivor, we investigated possible sources of the typical blood clotting. The results suggest that coagulation is probably initiated in a bimodal manner by an extracellular serine proteinase of the fungus and by elastase from the patients’ leukocytes. The former causes a partial hydrolysis of fibrinogen, while the latter activates coagulation factor XIII (fibrin stabilizing factor). Both proteinases were present in the patient at the site of infection, and in vitro they jointly bring about regular clotting of fibrinogen.
Ultrasonography and Cystic Hyperplasia–Pyometra Complex in the Bitch
Authors: Bigliardi E , Parmigiani E , Cavirani S , Luppi A , Bonati L , Corradi A
Published: June 2004

Contents

Cystic endometrial hyperplasia–pyometra complex is the most frequent and important endometrial disorder encountered in bitches. The pathogenesis of the disease is related to the activity of progesterone [Feldman and Nelson, Canine and Feline Endocrinology and Reproduction (1996) W.B. Saunders, Philadelphia]. Cystic endometrial hyperplasia (CEH) is an abnormal response of the bitch's uterus to ovarian hormones [De Bosschere et al. Theriogenology (2001) 55, 1509]. CEH is considered by many authors to be an exaggerated response of the uterus to chronic progestational stimulation during the luteal phase of the oestrous cycle, causing an abnormal accumulation of fluid within the endometrial glands and uterine lumen (De Bosschere et al. 2001). The resulting lesions of pyometra are due to the interaction between bacteria and hormones. The aim of this study was to evaluate if transabdominal uterine ultrasonography can be a useful and reliable diagnostic method to confirm Dow's [Veterinary Record (1958) 70, 1102] and De Bosschere's histopathological classification of CEH–pyometra complex. The study was carried out on 45 bitches with pyometra, 10 purebreeds and 35 crossbreeds, 1–15 years old, 20% of which had whelped at least once. None of these animals had received exogenous oestrogen or progesterone treatment. On admission the 45 animals were in the luteal phase of the oestrus cycle. Clinical signs, blood parameters, uterine ultrasonography, bacterial swabs and uterine histopalogical results were recorded. Results suggest that ultrasonographic examination is a useful and reliable tool for the diagnosis of cystic endometrial hyperplasia.
Evidence of Damage in Cryopreserved and Fresh Bovine Embryos Using the Tunel Technique
Authors: Márquez‐Alvarado YC , Galina CS , Castilla B , León H , Moreno‐Mendoza N
Published: June 2004

Contents

The objective of the present study was to evaluate the quality of bovine embryos cryopreserved in different years in Chiapas, México. The embryos were obtained from a government institution (FIMEGEN) dedicated to promoting embryo transfer among dual‐purpose cattle farmers. Forty‐three embryos frozen in 1988, 1989, 2000 and 2002 were analysed with the Tunel technique to detect programmed cell death (apoptosis). Eleven fresh embryos were used as controls. Analysis of variance was used in embryos stored in the different years with averages tested using Tukey's test. Student's t‐test was employed to compare fresh and frozen cells. Embryos with shorter storage time presented a lower number (p < 0.001) of Tunel‐positive cells compared with embryos stored for longer time. On the contrary, when comparing the number of apoptotic cells between frozen and fresh embryos a higher number of positive cells (p < 0.05) were found in the former. The present results suggest that the cryopreservation per se caused damage that compromises the viability of the embryo. Another explanation for the lower pregnancy rate found in the tropics could be irreversible damage caused by poor storage technique in these large operations.
Influences of Follicular Size on Parthenogenetic Activation and in Vitro Heat Shock on the Cytoskeleton in Cattle Oocytes
Authors: Tseng JK , Chen CH , Chou PC , Yeh SP , Ju JC
Published: June 2004

Contents

The availability of cow ovaries from the slaughterhouse has been very limited in Taiwan. To maximize the use of cow ovaries for research purposes, whole ovary dissection was performed and the developmental competence of the oocytes derived from different sizes of follicles was assessed by the rates of in vitro maturation (IVM) and parthenogenetic activation of the oocytes in Experiment 1 (Exp 1). Cumulus–oocyte complexes (COCs) derived from small (1–2 mm) and large (3–8 mm) follicles were subjected to standard IVM culture for 24 h. Mature oocytes were selected and then parthenogenetically activated using A23187 (5 μm, 5 min) or thimerosal (200 μm, 10 min) alone or combined with 6‐dimethylaminopurine (2.5 mm and 3.5 h, respectively). Activation rates of the oocytes, neither from the large nor small follicles, were affected by different activation treatments (single or combined stimuli). Whereas maturation rates for the oocytes from large follicles were superior to those from small follicles in both the single (59% vs 45%) and combined treatments (76% vs 40%; p < 0.05). To understand how prolonged heat shock (HS) influences cytoskeletal configurations of mature bovine oocytes, in Experiment 2 (Exp 2), matured oocytes derived from large follicles were randomly allocated to different durations of HS treatments at 41.5°C for 0 (C0h, control, n = 12), 1 (HS1h, n = 28), 2 (HS2h, n = 31), and 4 h (HS4h, n = 30). An additional control group was cultured for 4 h without HS (38.5°C, 4 h, n = 35). Alterations in nuclear structures, microtubules (MTs), and microfilaments (MFs) of the oocytes were examined. Abnormalities in the chromosomes, spindle MTs and the percentages of oocytes with cytoplasmic MTs increased with time of HS treatment. The intensity of the MF distribution in the HS oocytes was also altered. Significant changes in the cytoskeleton after HS may be associated with the reduced development under hyperthermia and, perhaps, with the low pregnancy rates of the animals during hot seasons.
Telomerase Activity in Swamp Buffalo (Bubalus bubalis) Oocytes and Embryos Derived from In Vitro Fertilization, Somatic Cell Nuclear Transfer and Parthenogenetic Activation
Authors: Saikhun J , Sritanaudomchai H , Pavasuthipaisit K , Kitiyanant Y
Published: June 2004

Contents

The objective of this study was to examine the telomerase activity in swamp buffalo oocytes and pre‐implantation stage embryos derived from in vitro fertilization (IVF), somatic cell nuclear transfer (NT) and parthenogenetic activation (PA). Immature and mature oocytes, and embryos at the 2–4 cell, 8–16 cell, morula and blastocyst stages produced by IVF, NT and PA were collected and the telomerase activity was assayed by using a Telomerase PCR ELISA kit. Telomerase activity was detected in all developmental stages evaluated from immature oocytes to blastocyst stage embryos. Telomerase activity was detected in higher amounts in immature as compared with mature oocytes (p < 0.05). Embryos derived from NT showed a profile of telomerase activity similar to that of IVF. In IVF and NT embryos, telomerase activity was low in the 2–4 cell and 8–16 cell stages, but the activity significantly increased (p < 0.05) at the morula stage, reaching its highest level at the blastocyst stage. In PA embryos, low levels of telomerase activity were detected from the 2–4 cell to the morula stage, and the highest level of telomerase activity was found at the blastocyst stage. Telomerase activity in NT blastocysts is higher than that derived from IVF and the activity is highest in PA blastocysts. These results suggest that the successful reprogramming of telomerase activity in buffalo NT embryos follow a pattern similar to that in embryos derived from IVF and PA.
Age‐related Variation of Plasma Concentrations of Skatole, Androstenone, Testosterone, Oestradiol‐17β, Oestrone Sulphate, Dehydroepiandrosterone Sulphate, Triiodothyronine and IGF‐1 in Six Entire Male Pigs
Authors: Zamaratskaia G , Babol J , Madej A , Squires EJ , Lundström K
Published: June 2004

Contents

This study describes the age‐related variation in boar taint compounds, skatole and androstenone, and testosterone, oestradiol‐17β (E17β), oestrone sulphate (ES), dehydroepiandrosterone sulphate (DHEAS), triiodothyronine (T3) and insulin‐like growth factor‐1 (IGF‐1) in six boars. Three pairs of littermates of crossbred entire male pigs (from three Yorkshire × Duroc dams and one Hampshire sire) were included. Blood samples were taken at the age of 9–15 weeks and thereafter at weekly intervals from the age of 20–32 weeks. Plasma concentrations of skatole, androstenone, testosterone, E17β, ES, DHEAS, T3 and IGF‐1 were measured. We found that skatole levels in boars increased at the age around puberty after an increase in the levels of testicular steroids. Levels of skatole were not associated with the levels of sex steroids, T3 and IGF‐1. However, the increased level of testicular steroids is probably the underlying factor needed for high skatole levels to occur although the specific mechanism leading to increased skatole levels remains unknown.
Characterization and Localization of Membrane Vesicles in Ejaculate Fractions from the Ram, Boar and Stallion
Authors: El‐Hajj Ghaoui R , Thomson PC , Evans G , Maxwell WMC
Published: June 2004

Contents

Membrane vesicles, separated by differential centrifugation from the seminal plasma, were detected in the sperm‐rich ejaculate fractions of four boars and three stallions, and in the whole ejaculates of seven rams. The volume and percentage of vesicles, determined by a stereological technique, were higher in the sperm‐rich than in the post‐sperm‐rich fractions of the boar and stallion ejaculates, and no vesicles were detected in the pre sperm‐rich fractions. Vesicles were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For boar, stallion and ram semen, the mean (± s.e.m.) vesicle diameters were 130.9 ± 3.22 (range 18–577), 164.1 ± 4.42 (range 15–671) and 159.7 ± 2.92 nm (range 22–986), respectively, although they were not significantly different (p = 0.709). The vesicles had approximately round (TEM) or spherical shape (SEM), were surrounded by single, double or multi‐laminar membranes, and were trapped within ample amorphous material, sometimes containing short, flattened membranous elements. The majority of the vesicles had a clear interior but some contained granule‐dense material. Ram membrane vesicles, purified from ultracentrifuged plasma by size exclusion chromatography, kept their round shape and the amorphous material was less evident compared with the sections taken before purification. This is the first report to identify seminal plasma membrane vesicles in the different fractions of ejaculated semen in the boar and stallion, and confirms their presence in ram seminal plasma. The origin and function of these vesicles are yet to be elucidated.
Effect of Gestational Age and Fetal Position on the Possibility and Accuracy of Ultrasonographic Fetal Gender Determination in Dairy Cattle
Authors: Ali A
Published: June 2004

Contents

The possibility and accuracy of transrectal ultrasonographic determination of foetal gender were studied by identifying and locating the genital tubercle, scrotum or mammary gland in 15 dairy cows at 49–168 days post‐breeding. Ultrasound examinations were performed weekly. Each examination was made without references to the results of the examinations in the previous weeks. At each examination, an attempt was made to view the foetus in frontal, cross‐sectional, and sagittal planes. The expected foetal gender was compared with that observed at birth. Gestational age affected significantly the possibility of foetal gender determination. The possibility of foetal gender determination increased from 26.7% at day 49 to 100% at day 56 of gestation. Between days 56 and 98, foetal gender determination was possible in all cases. Thereafter, the ability to determine foetal gender decreased with development of pregnancy and was impossible in all cases by days 161 and 168. The overall accuracy of foetal gender determination was 97.3%. Foetal gender determination between days 49 and 63 depended on the location of the genital tubercle in a frontal view of the foetus. By day 70, all foetal positions could be used for foetal gender determination. However, by progress of gestation, the usefulness of frontal view for sex determination decreased, while that of cross‐sectional view increased. Accordingly, the best time for foetal gender determination in dairy cattle is between days 56 and 98 of pregnancy.
Intra‐uterine Insemination in Farm Animals and Humans
Authors: Verberckmoes S , Van Soom A , de Kruif A
Published: June 2004

Contents

Artificial insemination (AI) is the oldest and currently most common technique in the assisted reproduction of animals and humans. The introduction of AI in farm animals was forced by sanitary reasons and the first large‐scale applications with a commercial goal were performed in cattle in the late 1930s of last century. After the Second World War, cryopreservation of semen facilitated distribution and AI was mainly performed for economic reasons, especially in dairy cattle industry. In humans however, AI was initially performed in cases of physiological and psychological sexual dysfunction, but later on also in cases of infertility caused by immunological problems. Currently, the most common indications for intra‐uterine insemination (IUI) in humans are unexplained infertility and male subfertility. In these cases, IUI is considered as the treatment of the first choice, before more invasive techniques such asin vitrofertilization (IVF) and intracytoplasmatic sperm injection (ICSI) are used. In contrast with humans, the quantity and quality of semen produced by farm animals is much higher and permits dilution and production of several insemination doses per ejaculate. However, with the introduction of sex‐sorted semen in farm animals, the same problem of low‐quality semen as in humans has arisen. In cattle, pigs and horses, conventional insemination with low numbers of sex‐sorted spermatozoa results in a significant decrease in fertility. To improve the fertility rates with this semen, new insemination techniques have been developed in order to deposit spermatozoa closer to the site of fertilization. In sows and mares the advantage of utero‐tubal junction (UTJ) insemination has already been proven; however, in cattle it is still under investigation. In this review, the differences and similarities in the application of AI between animals and humans are discussed and as AI in farm animals is most successful in cattle, the situation in this species is elaborated the most.
Apolipoprotein and anticardiolipin antibodies in patients with renal amyloidosis
Authors: CENGIZ KUDDUSI , KILIÇ MURAT , BEDIR ABDULKERIM
Published: June 2004

Background:  Thromboembolic events are seen more frequently in patients with chronic renal failure (CRF) and amyloidosis. The anticardiolipin antibody (ACA) that is important for thromboembolic events has never been studied.

Methods:  This study included 43 amyloidosis patients of different aetiologies; 28 with CRF as well as 20 patients who had CRF without amyloidosis. Thirty normal subjects were included as a control group. We determined the serum levels of ACA, apolipoprotein AI (ApoAl), ApoE and lipoprotein (a) (Lp(a)) in these groups.

Results:  Anticardiolipin antibody was found to be positive in 30.2% of patients with amyloidosis, this is in contrast to 3.3% in the control group (χ = 8.25, P < 0.005). We also showed that there was a statistically significant difference (χ = 5.03, P < 0.05) between the CRF patients with amyloidosis (31%) and the CRF patients without amyloidosis (5%). The average levels of serum ApoAI were shown to be significantly lower (P < 0.05) in CRF patients with amyloidosis in comparison with the amyloidosis patients who had normal renal functions (93.60 ± 27.84 vs 119.8 ± 36.26 mg/dL, P < 0.05). There was also significant a difference in ApoAI levels between CRF patients with and without amyloidosis (P < 0.001). The serum Lp (a) levels were significantly higher in CRF patients with amyloidosis when compared with the controls (41.2 ± 22.39 vs 19.13 ± 8.78 mg/dL, P < 0.001). The serum Lp (a) levels were also positively correlated with ACA (r = 0.211; P < 0.05).

Conclusion:  In conclusion, ACA positivity is more common in all patients with amyloidosis as compared with CRF patients and normal controls. This study is the first to show the presence of high levels of ACA in patients with CRF, which is caused by secondary amyloidosis. Further studies are recommended to investigate the mechanism of this finding.
Atherosclerosis and folic acid supplementation trial in chronic renal failure: Baseline results
Authors: ZOUNGAS SOPHIA , BRANLEY PAULINE , KERR PETER G , RISTEVSKI SONYA , MUSKE CHRISTINE , DEMOS LISA , ATKINS ROBERT C , BECKER GAVIN , FRAENKEL MARGARET , HUTCHISON BRIAN G , WALKER ROBERT , MCNEIL JOHN J , MCGRATH BARRY P
Published: June 2004

Background:  Atherosclerosis and Folic Acid Supplementation Trial (ASFAST) is a randomized placebo controlled trial assessing whether high‐dose folic acid can reduce cardiovascular events and atherosclerosis progression in patients with chronic renal failure (CRF). Here we report the baseline results and compare indices of arterial structure (carotid intima‐medial thickness (IMT)) and function (systemic arterial compliance (SAC)), pressure augmentation index (AIx) and pulse wave velocity (PWV a‐f and PWV f‐d)) to age‐ and sex‐matched controls.

Methods:  Three hundred and fifteen subjects with CRF (serum creatinine ≥ 0.40 mmol/L) aged 24–79 years (mean ± SD: 56.6 ± 13.6 years) and 213 healthy controls (58.2 ± 10.2 years) were studied. Fasting blood samples were assayed for lipids (both groups), total homocysteine (tHcy), red cell folate, cobalamin and fibrinogen (CRF group). Ultrasound B mode measurements were used to determine mean  carotid  IMT  and  applanation  tonometry  techniques  to  determine  SAC,  AIx,  PWV  (a‐f),  PWV (f‐d) and central pressures.

Results:  Ninety‐six per cent of the CRF group had at least one of: hypertension, hypercholesterolaemia, diabetes or smoking; 35% had established cardiovascular disease. The mean IMT was greater in CRF patients than in controls (0.86 ± 0.19 vs 0.68 ± 0.11 mm, P < 0.001). The SAC was significantly lower, and PWV (a‐f) and AIx significantly higher. The tHcy was increased in 97% of the CRF group (27.3 ± 2.9 µmol/L (normal < 13)). Total homocysteine did not correlate with IMT or any other measure of arterial function. However, those in the upper quantile of tHcy (≥25 µmol/L) did have higher PWV (a‐f) and lower SAC than those in the lower quantile.

Conclusions:  Compared to normals, patients with CRF exhibited a 10–15‐year shift to the right in age‐related increases in carotid IMT and PWV (a‐f), and significantly increased central pressure augmentation. This 5‐year study is examining the impact of high‐dose folic acid therapy on cardiovascular end‐points, IMT progression and arterial function in CRF.
Antibiotic prophylaxis in dialysis patients undergoing invasive dental treatment
Authors: TONG DARRYL C , WALKER ROBERT J
Published: June 2004

Background and Aims:  The role of antibiotic prophylaxis for invasive dental procedures in patients on dialysis therapy is unclear. We examined current clinical practice in Australia and New Zealand and compared our findings to a systematic review of the current literature.

Methods::  Australian and New Zealand nephrology units were surveyed with regard to their use of antibiotic prophylaxis for dental procedures. A systematic review of the literature was performed by using an online web‐based search engine (PubMed ®) using the key words: renal patients, dental and antibiotic prophylaxis.

Results:  Forty‐one per cent of respondents do not routinely give antibiotic prophylaxis to haemodialysis patients prior to dental surgery, but a majority (53%) would consider antibiotic prophylaxis if the patient had a synthetic arteriovenous fistula.

Conclusions:  The majority of clinicians follow the American Heart Association (AHA) guidelines with a single oral preoperative dose of 2 g amoxycillin or 600 mg clindamycin if patients are allergic to penicillin. From the literature and the data obtained by questionnaire, it would appear that renal patients receiving haemodialysis in Australia and New Zealand receive antibiotic prophylaxis prior to invasive dental procedures. The standard single dose of 2 g amoxycillin orally or 600 mg clindamycin orally 1 h preoperatively, as recommended by the AHA, is most frequently used. Peritoneal dialysis patients generally do not receive a prophylactic dose of antibiotics.
A case of primary immunoglobulin light chain amyloidosis with a delayed appearance of Bence Jones protein in urine
Authors: KURUSU ATSUSHI , YAMADA TOSHIYUKI , YAMAJI KENJI , NISHITANI MIHO , TASHIRO KYOICHI , MAEDA KUNIMI , HORIKOSHI SATOSHI , SHIRATO ISAO , RINNO HISAKI , TOMINO YASUHIKO
Published: June 2004

SUMMARY:  We report here a case of a 58‐year‐old man who had nephrotic syndrome and immunoglobulin light chain (AL) amyloidosis. This patient underwent a renal biopsy to confirm the diagnosis. Treatment with permanganate before Congo red staining showed systemic secondary amyloidosis (AA) fibrils, which were sensitive to permanganate oxidation. Although this patient was initially diagnosed as having AA amyloidosis, he did not have any chronic inflammatory disease and/or malignancy. The level of amyloid A protein (7.9 µg/mL) in sera was within the normal range (0–8.0 µg/mL). Therefore, we performed an immunostaining of the precursor protein (amino terminus of constant region: κ and λ light chains, and AA protein) using duodenal biopsy specimens for a precise diagnosis. Immunostaining was positive for the amino terminus of constant region of the λ light chain, and negative for the amino terminus of constant region of the κ light chain and AA protein. No plasma cell proliferation in the bone marrow was observed. We finally diagnosed this patient as having primary AL amyloidosis. It appears that a pathological diagnosis must be performed by immunostaining the precursor proteins with the permanganate digestion technique in tissue of patients with amyloidosis. There were no abnormalities in serum and urine immunoelectrophoresis at the time of renal biopsy in this patient. During the follow‐up period, after discharge, Bence Jones protein appeared in the urine, but not in the serum. It is necessary to observe patients with primary AL amyloidosis carefully to determine if they their condition will progress to multiple myeloma.
Renoprotective effect of Hemidesmus indicus, a herbal drug used in gentamicin‐induced renal toxicity
Authors: KOTNIS MANGALA S , PATEL PRATEEK , MENON SASHIKUMAR NARAYAN , SANE RAMESH TRIMBAK
Published: June 2004

Background and Aims:  Owing to the global trend towards improved ‘quality of life’, there is considerable evidence of an increase in demand for medicinal plants. The WHO guidelines define basic criteria for the standardization of herbal medicines. The present work is an effort in this direction to prove the safety and efficacy of Hemidesmus indicus Linn. in the management of nephrotoxicity induced by aminoglycosides such as gentamicin.

Methods and Results:  Simple, quality control methods using high performance thin layer chromatographic (HPTLC) phytochemical fingerprint, proximate analysis, and the stability of the H. indicus root powder were developed. From the toxicity study using albino Swiss mice, it was observed that the drug (H. indicus) was relatively safe up to 7 g/kg bodyweight dose. Efficacy was evaluated against gentamicin‐induced nephrotoxicity in albino Wister rats. The study examined animals from the following groups: no treatment, gentamicin treated, gentamicin treated recovery, and gentamicin and plant treated. Animals from all groups were killed on day 13 of the study; those from gentamicin treated group were killed on the seventh day. Assessment of the drug efficacy drug was conducted by using haematological and histological examination.

Conclusion:  The treatment with H. indicus helped in the management of renal impairment, which was induced by gentamicin in rats. This is evident from the results obtained for various kidney function tests for gentamicin, along with the results from the plant treated group, and is in comparison with the results found for the gentamicin recovery group. A histological examination of kidneys also supports the findings from haematological evaluations. The plant shows promise as an adjunct therapy along side aminoglycosides as it reduces nephrotoxicity caused by aminoglycosides.
Relationship of predialytic intact parathyroid hormone on secondary hyperparathyroidism in chronic maintenance haemodialysis patients
Authors: ETOH SATOSHI , MURATA TOSHIAKI , HASEGAWA YOSHIYUKI , MIYAHARA YOSHITO , ISHIMURA ATUNORI , ABE YASUHIRO , NODA RITSUYA , OGAHARA SATORU , KANEOKA HIDETOSHI , SAITO TAKAO
Published: June 2004

Methods and Results:  In order to clarify the predialytic factors influencing the onset of secondary hyperparathyroidism (SHPT) in patients on chronic maintenance haemodialysis, the time‐course changes of serum levels of intact‐PTH (i‐PTH) during haemodialysis for 5 years were investigated. The subjects were 69 non‐diabetic patients who had a serum aluminium level of less than 1.85 nmol/L at the end of observation. Patients were divided into two groups based on i‐PTH levels obtained at the start of dialysis; the high group (H group) consisted of patients whose i‐PTH levels were more than 22.00 pmol/L, the low group (L group) had levels less than 22.00 pmol/L. In the H group, i‐PTH was 41.46 ± 2.87 pmol/L at the start of dialysis (vs L group, P < 0.0001) and 15.82 ± 2.85 pmol/L after haemodialysis initiation. In the L group, i‐PTH levels did not significantly change and was 11.69 ± 2.50 pmol/L 12 months after the start of dialysis (at the 12th month). However, at the 60th month, the i‐PTH level was 33.24 ± 5.30 pmol/L in the H group, and 9.85 ± 2.13 pmol/L in the L group (P < 0.005).

Conclusion:  It is suggested that control of i‐PTH levels in the predialytic period may be important to suppress SHPT throughout haemodialysis.