The influence of litter birth weight phenotype on embryonic and placental development at day 30 of gestation in multiparous purebred Large White sows.

The aim of this study was to understand the intrauterine biological processes associated with the low litter birth weight phenotype in pigs. Analyses were conducted on reproductive data from a purebred Large White maternal line to identify sows (>2 parities) with repeatable high or low litter birth weight phenotype (HLBWP or LLBWP). A total of 40 sows were selected (n = 20 HLBWP and n = 20 LLBWP) and bred with semen from purebred Large White boars of proven fertility. Sows were euthanized on day 28-30 of gestation (day 29.5 ± 0.6) and samples of placenta and embryos collected. Total number of embryos (TNE), embryonic weight (EW), embryonic viability, and crown-rump (CRL) measurements were recorded, along with the ovulation rate (OR) and allantochorionic fluid volume (AFV). No significant difference was detected (P > 0.05) in OR, TNE, and number of viable embryos on day 30 of gestation between the two groups. There was no significant difference in EW (LLBWP: 0.80 ± 0.05 g; HLBWP: 0.88 ± 0.04 g, P = 0.18) or CRL (LLBWP: 21.5 ± 0.7 mm; HLBWP: 21.9 ± 0.68 mm, P = 0.46). Placental development represented by the average AFV was significantly lower in the LLBWP compared to HLBWP (LLBWP: 131 ± 9.82 mL; HLBWP: 149 ± 9.39 mL, P = 0.03). In conclusion, placental development may be the main factor causing lower BW of entire litters in LLBWP sows.

Validation of an automated colony counting system for group A Streptococcus.

BACKGROUND: The practice of counting bacterial colony forming units on agar plates has long been used as a method to estimate the concentration of live bacteria in culture. However, due to the laborious and potentially error prone nature of this measurement technique, an alternative method is desirable. Recent technologic advancements have facilitated the development of automated colony counting systems, which reduce errors introduced during the manual counting process and recording of information. An additional benefit is the significant reduction in time taken to analyse colony counting data. Whilst automated counting procedures have been validated for a number of microorganisms, the process has not been successful for all bacteria due to the requirement for a relatively high contrast between bacterial colonies and growth medium. The purpose of this study was to validate an automated counting system for use with group A Streptococcus (GAS). METHODS: Twenty-one different GAS strains, representative of major emm-types, were selected for assessment. In order to introduce the required contrast for automated counting, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) dye was added to Todd-Hewitt broth with yeast extract (THY) agar. Growth on THY agar with TTC was compared with growth on blood agar and THY agar to ensure the dye was not detrimental to bacterial growth. Automated colony counts using a ProtoCOL 3 instrument were compared with manual counting to confirm accuracy over the stages of the growth cycle (latent, mid-log and stationary phases) and in a number of different assays. The average percentage differences between plating and counting methods were analysed using the Bland-Altman method. RESULTS: A percentage difference of ±10 % was determined as the cut-off for a critical difference between plating and counting methods. All strains measured had an average difference of less than 10 % when plated on THY agar with TTC. This consistency was also observed over all phases of the growth cycle and when plated in blood following bactericidal assays. Agreement between these methods suggest the use of an automated colony counting technique for GAS will significantly reduce time spent counting bacteria to enable a more efficient and accurate measurement of bacteria concentration in culture.

Gene expression of porcine blastocysts from gilts fed organic or inorganic selenium and pyridoxine.

In this study, we determined how maternal dietary supplementation with pyridoxine combined with different sources of selenium (Se) affected global gene expression of porcine expanded blastocysts (PEB) during pregnancy. Eighteen gilts were randomly assigned to one of the three experimental diets (n=6 per treatment): i) basal diet without supplemental Se or pyridoxine (CONT); ii) CONT+0.3 mg/kg of Na-selenite and 10 mg/kg of HCl-pyridoxine (MSeB610); and iii) CONT+0.3 mg/kg of Se-enriched yeast and 10 mg/kg of HCl-pyridoxine (OSeB610). All gilts were inseminated at their fifth post-pubertal estrus and killed 5 days later for embryo harvesting. A porcine embryo-specific microarray was used to detect differentially gene expression between MSeB610 vs CONT, OSeB610 vs CONT, and OSeB610 vs MSeB610. CONT gilts had lower whole blood Se and erythrocyte pyridoxal-5-P concentrations than supplemented gilts (P<0.05). No treatment effect was observed on blood plasma Se-glutathione peroxidase activity (P=0.57). There were 10, 247, and 96 differentially expressed genes for MSeB610 vs CONT, OSeB610 vs CONT, and OSeB610 vs MSeB610 respectively. No specific biological process was associated with MSeB610 vs CONT. However, for OSeB610 vs CONT, upregulated genes were related with global protein synthesis but not to selenoproteins. The stimulation of some genes related with monooxygenase and thioredoxin families was confirmed by quantitative real-time RT-PCR. In conclusion, OSeB610 affects PEB metabolism more markedly than MSeB610. Neither Se sources with pyridoxine influenced the Se-glutathione peroxidase metabolic pathway in the PEB, but OSeB610 selectively stimulated genes involved with antioxidant defense.

Reproductive technologies and the porcine embryonic transcriptome.

The domestic pig is not only an economically-important livestock species, but also an increasingly recognized biomedical animal model due to its physiological similarities with humans. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in the pig using either in vivo or in vitro production methods. The application of assisted reproductive technologies (ART) has the potential to increase reproductive efficiency in livestock. These technologies include, but are not limited to: artificial insemination (AI), fixed-time AI, embryo transfer, cryopreservation of sperm/oocytes/embryos, in vitro fertilization and somatic cell nuclear transfer (cloning). However, the application of ART is much less efficient in the pig than in many other mammalian species such as cattle. Until recently, the underlying causes of these inefficiencies have been difficult to study, but advances in molecular biology techniques for studying gene expression have resulted in the availability of a variety of options for gene expression profiling such as microarrays, and next generation sequencing technologies. Capitalizing on these technologies the effects of various ARTs on the porcine embryonic transcriptome has been determined and the impact on the related biological pathways and functions been evaluated. The implications of these results on the efficiency of ARTs in swine, as well potential consequences for the developing embryo and resulting offspring, are reviewed.

Hypoxia and lactate production in trophoblast cells.

The etiology of preeclampsia is unknown but is thought to be related to hypoxia in the placenta. We previously reported that the enzyme lactate dehydrogenase (LDH) has increased activity and gene expression in placentas from preeclamptic pregnancies [Tsoi SCM, Zheng J, Xu F, Kay HH. Differential expression of lactate dehydrogenase isozymes (LDH) in human placenta with high expression of LDH-A(4) isozyme in the endothelial cells of pre-eclampsia villi. Placenta 2001;22:317-22]. LDH is responsible for pyruvate conversion to lactate through glycolysis. In this study, we further investigated the role of hypoxia in primary trophoblast cells and a cultured cell line, JEG3 cells, to obtain a better understanding of how it affects the activities of lactate dehydrogenase, lactate production and regulatory genes, as a possible model for preeclampsia. Primary trophoblast cells and JEG3 cells were cultured under 1% oxygen. At 6, 12 and 24h, cells were analyzed for LDHA and LDHB isozyme activities, mRNA and protein expression compared to standard culture conditions. Lactate was measured from cell medium. The hypoxia inducible transcription factor (HIF-1alpha) protein expression was confirmed by western blot. Two lactate transporters (MCT1 and MCT4) mRNA and protein expression were also studied under hypoxia. Finally, lactate was measured in plasma obtained from patients with severe preeclampsia. Under hypoxic conditions, LDHA mRNA is increased in primary trophoblast cells and JEG3 cells. The HIF-1alpha protein expression is higher in hypoxia-treated JEG3 cells than control. LDHA isozyme activity and its protein expression are increased most significantly at 24h of culture under hypoxia. However, LDHB protein is unchanged while its mRNA is decreased. Lactate secretion from JEG3 cells under hypoxia is increased, as is the lactate levels in the plasma from preeclampsia patients. Of the two lactate transporters studied, MCT4 mRNA and protein level are increased under hypoxia. Our findings support the role of hypoxia in inducing HIF-1alpha activity in trophoblasts and increasing LDH transcription as well as its activity. Higher levels of lactate are produced and secreted which may contribute to the higher lactate levels in plasma of preeclamptic patients. These mechanisms may be important in the pathophysiology of preeclampsia.

Identification of immune-relevant genes from atlantic salmon using suppression subtractive hybridization.

In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon (Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process. Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314-BQ037059). Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries. These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others. A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription-polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection.

[Severe parkinsonism due to metoclopramide in a patient with polypharmacy]. / Ernstig parkinsonisme ten gevolge van metoclopramide bij een patiënte met polyfarmacie.

A 73-year-old woman, with tuberculosis of the large intestine, developed nausea as a side effect of the antituberculosis drugs. The nausea was treated with metoclopramide. Subsequently she developed severe medication-induced parkinsonism. As her symptoms initially mimicked a depressive disorder, drug-induced parkinsonism was only considered at a later stage. Due to drug-induced impaired function of the liver and kidney the patient had received a toxic dose of metoclopramide. Treatment with biperiden and withdrawal of the metoclopramide resulted in a reduction of the complaints within 3 months, after which the anti-tuberculosis medication could be reintroduced. Adjusting the dose of metoclopramide could possibly have prevented this severe side effect.

Differential expression of lactate dehydrogenase isozymes (LDH) in human placenta with high expression of LDH-A(4) isozyme in the endothelial cells of pre-eclampsia villi.

To evaluate the role of LDH isozymes in the human placenta during the third trimester, placentae were obtained from patients with normal pregnancy and pre-eclampsia. LDH-A(4)isozyme was immunolocalized primarily in the fetal endothelial cells while LDH-B(4)isozyme was predominantly present in syncytiotrophoblasts. This distinct cellular expression pattern of LDH isozymes was confirmed in HUVE and JEG cells. In addition to demonstrating the presence of five LDH isozymes in the placenta, zymograms showed that there was predominant activity of LDH-A(4)isozyme in HUVE cells and high activity of LDH-B(4)in JEG cells. Quantitative studies of LDH by agarose gel electrophoresis and Northern analysis in patients concluded that LDH-A(4)isozyme was increased in pre-eclampsia. The LDH-A(4)isozyme activity increased (P< 0.01) approx 1.6-fold in pre-eclampsia but there was no difference in the LDH-B(4)isozyme activity between placentae from normal compared to pre-eclampsia pregnancy. The level of LDH-A mRNA was increased (P< 0.05) approx twofold in pre-eclampsia. We conclude that the LDH-A gene in the endothelial cells of the placenta within the fetal microvasculature is increased in pre-eclampsia, probably as a result of hypoxia. LDH-A(4)isozyme activity and gene expression in placental endothelial cells, therefore, is a marker for the endothelial pathology seen in pre-eclampsia.

Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster.

A SMT3 cDNA encoding ubiquitin-like protein from Drosophila melanogaster was isolated and sequenced. Drosophila SMT3 genomic DNA was amplified by polymerase chain reaction, and its nucleotide sequence was found to be identical to that of the cDNA, indicating the absence of intron in its protein coding region. The sequence of 90 amino acids of Drosophila SMT3 exhibited 55%, 73%, 70% and 52% identity to yeast SMT3, human SMT3A, SMT3B and SMT3C protein.

cDNA cloning and genomic organization of enhancer of split groucho gene from nematode Caenorhabditis elegans.

This first genomic Enhancer of split groucho (ESG) gene and its full length complementary DNA (cDNA) from nematode C. elegans were cloned and sequenced via homology with the corresponding Drosophila groucho cDNA. The cDNA of 2.1-Kb encodes a protein of 612 amino acids, and the nematode ESG protein is the smallest and most different in structure compared to all ESG related proteins. The gene isolated is 4,246-bp in size, including 1,219-bp promoter region. A putative TATA-box at position -1166, two consensus sequence of ACTGG, characteristic of leader binding protein-1 (LBP-1) binding motifs at position -563 and -211 and nine CAAT boxes were found in the promoter region of ESG gene. The protein-coding sequence is interrupted by five introns. The length of introns 1 to 5 is 52, 252, 87, 53 and 518 bp, respectively. The overall structural relationships of the ESG-related proteins among human, mouse, rat, Xenopus, Drosophila and nematode were also analyzed.

The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes.

The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.

Sequences of the lizard cDNAs encoding lactate dehydrogenase (LDH) isozymes A (muscle) and B (heart).

The nucleotide and deduced amino acid sequences of cDNAs encoding L-lactate dehydrogenase (LDH) isozymes A (muscle) and B (heart) from the lizard, Sceloporus undulatus, were determined. The evolutionary relationships among LDH isozymes from animals, plants and bacteria are presented.

The nucleotide and deduced amino-acid sequences of a cDNA encoding lactate dehydrogenase from Caenorhabditis elegans: the evolutionary relationships of lactate dehydrogenases from mammals, birds, amphibian, fish, nematode, plants, bacteria, mycoplasma, and plasmodium.

The nucleotide and deduced amino-acid sequences of a cDNA encoding L-lactate dehydrogenase (LDH) from nematode, Caenorhabditis elegans, were reported. This first invertebrate LDH sequence of 333 amino acids, including the initiation methionine, exhibits 63% identity with that of the most primitive vertebrate lamprey. The evolutionary relationships among 36 LDH isozymes from mammals, birds, amphibian, fish, nematode, plants, bacteria, mycoplasma and plasmodium were analyzed. The invertebrate nematode LDH is evolutionarily positioned between plant LDH and mammalian testicular LDH-C isozymes. The mammalian LDH-C isozyme appears to have arisen after the invertebrate LDH, but prior to the divergence of vertebrate LDH-A (muscle) and LDH-B (heart) isozymes as described previously.

Responses of frog vestibular neurons to combined temperature microstimulation of the semicircular canals.

The temperature microstimulation of the semicircular canal (heat bursts lasting about 2 sec with a peak amplitude of 0.5-5.0 degrees C) can be regarded as an analog of angular acceleration acting within the cavity of the given canal; the combination of temperature microstimulations targeted simultaneously on several canals can serve as a physical model of accelerated rotation having complex spatial characteristics. The recording of the activity of neurons of the vestibular nuclei of the frog (n = 278) in response to temperature microstimulation of the canals showed that 80% of the neurons have inputs from one to two canals and only 20% from three to six canals. The distribution of laterality was characterized by the predominance of ipsilateral inputs: 201 neurons (72.3%) had only ipsilateral inputs; 14 neurons (5%) had only contralateral inputs; 63 neurons (22.7%) had both inputs. The ipsilateral horizontal (67.6%) and the posterior (61.9%) were the most effective inputs; while among the contralateral, the posterior (21.2%) canals were the most effective; the least effective were the contralateral horizontal (8.6%) and the anterior (5.0%) canals. The presence of latent canal inputs (both excitatory and inhibitory) which showed up in combination with effective inputs was demonstrated.

[The responses of frog vestibular neurons to the combined temperature microstimulation of the semicircular canals]. / Otvety vestibuliarnykh neironov liagushki na kombinirovannuiu temperaturnuiu mikrostimuliatsiiu polukruzhnykh kanalov.

Controlled temperature microstimulation of the frog semicircular canal (heating of 2 sec duration, peak amplitude from 0.5 to 5.0 degrees C above the temperature level of the labyrinth, 17-19 degrees C) may be considered as an analogue of angular acceleration in the plane of the canal. Combined temperature microstimulation of some canals may be considered as a physical model of complicated space rotations. Responses of the frog vestibular nuclei's neurons (n = 278) to temperature microstimulation showed that 80% of them had inputs from 1-2 canals and only 20% of neurons--from 3-6 canals. 201 neurons (72.3%) had ipsilateral inputs only; 14 neurons (5%)--contralateral ones only; 63 neurons (22.7%) had both inputs. The most effective excitatory inputs were ipsilateral horizontal, ipsilateral posterior and contralateral posterior canals; the least effective were contralateral horizontal and contralateral anterior canals. Latent canal inputs (excitatory as well as inhibitory) seem to exist as revealed in combination with the effective inputs.

[The initial results of the sanatorium rehabilitation of patients having had an acute myocardial infarct in a mid-alpine climate]. / Pervye itogi sanatornoi reabilitatsii bol'nykh, perenesshikh ostryi infarkt miokarda, v usloviiakh srednegornogo klimata.

Short-term results of rehabilitation of 1046 CHD patients at a cardiological sanatorium in mountains at average altitude (1600 m above the sea level) were studied. Large focal myocardial infarction (MI) was found in 738 of them, small focal MI in 249, and acute focal myocardial dystrophy of ischemic genesis in 59. Of 1046 patients considerable improvement of the status after rehabilitation therapy at the sanatorium was noted in 27.6%, improvement in 64.5%, no changes in 6.1%, deterioration in 1.7%; one patient died. This type of therapy in a mountainous climate at average altitude yielded good immediate results in most of the patients (92.06%) with MI and could be recommended for rehabilitation of CHD patients at the 2nd stage of treatment at a sanatorium.

[Possible role of the potential-dependent potassium channels in the generation of responses of the pacinian corpuscles]. / Vozmozhnaia rol' potentsialozavisimykh kalievykh kanalov v generatsii otvetov telets Pachini.

Action of TEA (20 mM) and 4-AP (5 mM) on mechano- and electrically excitable membranes of the Pacinian corpuscles was studied by means of the air gap technique in constantly perfused preparations. Extracellular recordings of receptor and action potentials have shown that application of TEA causes prolongation of the receptor potential and inhibition of its amplitude by 40%. 4-AP has no effect on the mechanosensitive membrane. TEA and 4-AP induce a 2-3 fold increase of the action potential duration in the first Ranvier node. The computations based on the Dodge model help revealing that inhibition of voltage-sensitive potassium channels which participate in the first-order coding of the intact receptor accounts for this increase.