Glycogen phosphorylase: developmental expression in rat liver

We studied the superficial abdominal reflexes of 83 normal men, using as stimuli a train of electrical pulses or a needle scratch. Electrical stimulation delivered to the midline of the abdominal wall evoked, almost symmetrically on both sides, two reflex discharges: an early response having an oligophasic wave form, and a late response of polyphasic wave form. The threshold of the early response significantly exceeded that of the late response. With repetitive stimulation, the late response generally revealed habituation. Electrical stimulation of the unilateral abdominal wall evoked two responses on the stimulated side, whereas it evoked only the late response on the contralateral side. A needle scratch on the unilateral abdominal wall evoked one reflex discharge with a long latency and a polyphasic wave form. This response occurred generally on the stimulated side and became habituated to repeated scratching. These observations suggest that the superficial abdominal reflexes elicited by electrical stimulation are composed of two reflex discharges with a different reflex arc. They appear to closely resemble the blink reflex. The response elicited by needle scratching is thought to correspond to the late response of the electrically elicited abdominal reflexes.     

Neurokinin 1 receptor expression in the rat retina

Tachykinin (TK) peptides influence neuronal activity in the inner retina of mammals. The aim of this investigation was to determine the cellular localization of the neurokinin 1 receptor (NK1), whose preferred ligand is the TK peptide substance P (SP), in the rat retina. These studies used a polyclonal antiserum directed to the C-terminus of rat NK1. The majority of NK1-immunoreactive (IR) cells were located in the proximal inner nuclear layer (INL), and very rarely they were found in the distal INL. Some small and large NK1-IR somata were present in the ganglion cell layer. NK1-IR processes were densely distributed across the inner plexiform layer (IPL) with a maximum density over lamina 2 of the IPL. Immunoreactive processes also crossed the INL and ramified in the outer plexiform layer where they formed a sparse meshwork. NK1-IR processes were rarely observed in the optic nerve fiber layer. Double-label immunofluorescence studies with different histochemical markers for bipolar cells indicated that NK1 immunoreactivity was not present in bipolar cells. Together, these observations indicate that NK1 immunoreactivity is predominantly expressed by amacrine, displaced amacrine, interplexiform, and some ganglion cells. Double-label immunofluorescence experiments were also performed to characterize NK1-containing amacrine cells. Sixty-one percent of the gamma-aminobutyric acid (GABA)-IR cells, 71% of the large tyrosine hydroxylase (TH)-IR cells, and 100% of the small TH-IR cells contained NK1 immunoreactivity. In addition, most (91%) of the NK1-IR cells had GABA immunoreactivity. In contrast, vasoactive intestinal polypeptide-, TK-, choline acetyltransferase-, and parvalbumin-IR amacrine tells did not express NK1 immunoreactivity. Overall, the present findings suggest that SP acts directly upon several cell populations, including GABA-containing amacrine cells and ganglion cells, to influence visual information processing in the inner retina.     

Manipulation of metallothionein expression in the regenerating rat liver using antisense oligonucleotides

In order to gain some insight into the fate of fumarate synthesised in the cytosol in the purine nucleotide cycle and in amino acid catabolism, the capability of both rat kidney mitochondria and acidotic rat kidney mitochondria to take up either externally synthesised, via adenylsuccinate lyase, or added fumarate in exchange with intramitochondrial malate or aspartate was tested by means of both spectrophotometric and isotopic techniques. The appearance of either malate or aspartate caused by the presence of fumarate was revealed outside normal and acidotic mitochondria by using specific substrate detecting systems. Consistently, externally added fumarate was found to cause efflux of either [14C]-malate or [14C]-aspartate from loaded mitochondria. The occurrence in rat kidney mitochondria of two separate translocators, i.e., fumarate/malate and fumarate/aspartate carriers, is shown in the light of saturation kinetics and the different inhibitor sensitivity. The fumarate/aspartate antiporters found in normal and acidotic mitochondria appear to differ from each other.     

Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat

BACKGROUND & AIMS: Inflammatory stimuli and lipid peroxidation up-regulate cyclooxygenase (COX)-2. This study evaluated the relationship between inflammatory mediators, COX expression, and pathological changes in experimental alcoholic liver disease. METHODS: Rats (5 per group) were fed ethanol and a diet containing saturated fat, corn oil, or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in controls. In the first set of experiments, whole livers were analyzed. In the second set of experiments, Kupffer cells, endothelial cells, and hepatocytes were isolated from rats in each group. Pathological analyses and measurements of lipid peroxidation, tumor necrosis factor (TNF)-alpha, COX-1 and COX-2 messenger RNA (mRNA), endotoxin, and liver and plasma thromboxane were performed. RESULTS: Increased expression of COX-2 mRNA was detected in the livers of rats showing necroinflammatory changes. The Kupffer cell was the cell primarily responsible for the increase in COX-2 mRNA level. Increased expression of COX-2 was associated with increased levels of endotoxin, TNF-alpha mRNA, lipid peroxidation, and synthesis of thromboxane. COX-1 mRNA was decreased in Kupffer cells in rats with the most severe liver injury. CONCLUSIONS: Up-regulation of COX-2 in alcoholic liver injury occurred in the presence of proinflammatory stimuli and resulted in increased synthesis of inflammatory and vasoactive eicosanoids. Down-regulation of COX-1 may result in decreased synthesis of cytoprotective eicosanoids and additionally exacerbate liver injury.     

cDNA expression studies of rat liver aryl sulphotransferase

A cDNA encoding an isoenzyme of rat liver aryl sulphotransferase was isolated from a rat liver bacteriophage Lambda gt 11 library by the polymerase chain reaction technique. The resulting cDNA was functionally expressed in COS-7 cells and characterised by determining the sulphating capacity of the cells with a range of substrates. The COS-expressed enzyme catalysed the sulphation of both phenol and dopamine with Kms of the same order as those obtained for the high affinity isozyme in rat liver cytosol, while low activity was observed with tyrosine methyl ester. The common food additive vanillin was also a good substrate for sulphate conjugation. The sulphation of vanillin catalysed by the COS-expressed enzyme was consistent with a single enzyme system, in contrast, the kinetics of the reaction catalysed by cytosolic sulphotransferase indicated that vanillin was sulphated by more than one isozyme.     

Control of expression of the gene for the arginine transporter Cat-1 in rat liver cells by glucocorticoids and insulin

A general mathematical model for the dynamic behaviour of a single-compartment respiratory system in response to an arbitrary applied inspiratory airway pressure and arbitrary respiratory muscle activity is investigated. The model is used to compute explicit expressions for ventilation and pressure variables of clinical interest for clinician-selected and impedance-determined inputs. The outcome variables include tidal volume, end-expiratory pressure, minute ventilation, mean alveolar pressure, average pleural pressure, as well as the work performed by the ventilator and the respiratory muscles. It is also demonstrated that under suitable conditions, there is a flow reversal that can occur during inspiration.     

Gene expression of syndecans and betaglycan in isolated rat liver cells

Competence as communication skills and as skilled practice of asepsis were studied by observing four nurses while interacting with patients and performing intravenous procedures. Nurses were observed using sterile equipment for methods of intravenous therapy. Asepsis is performed frequently, but through misunderstanding in the learning of asepsis or improper model learning the nurses may establish incorrect routines. When performing procedures, unexpected factors can distract both the expert and the inexperienced, resulting in a failure to apply basic aseptic techniques. The nurses showed an interest in the patient by listening and giving responses. Nurses may control interactions with the patient by using undesirable communication skills which include incomplete sentences, incomplete explanations and closed questions. Asked to evaluate their own behavior, the nurses did not estimate whether or not the appropriate skills were applied in observed situations. To improve the quality of nursing care performance it is recommended to further develop and apply skill training programs.     

Growth-related changes in phosphorylation of yeast RNA polymerase II

The largest subunit of RNA polymerase II contains a unique C-terminal domain (CTD) consisting of tandem repeats of the consensus heptapeptide sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals.     

Decreased expression and activity of P-glycoprotein in rat liver during acute inflammation

PURPOSE: Drug disposition is often altered in inflammatory disease. Although the influence of inflammation on hepatic drug metabolism and protein binding has been well studied, its impact on drug transport has largely been overlooked. The multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) is involved in the active secretion of a large variety of drugs. Our goal was to ascertain the influence of acute inflammation (AI) on the expression and functional activity of P-gp. METHODS: AI was induced in rats through turpentine or lipopolysaccharide (LPS) administration. Expression of P-gp in liver was detected at the level of protein on Western blots using the monoclonal antibody C-219 and at the level of mRNA using an RNase protection assay. P-gp mediated transport activity was assessed by measuring the verapamil-inhibitable efflux of rhodamine 123 (R123) in freshly isolated hepatocytes. RESULTS: Turpentine-induced AI significantly decreased the hepatic protein expression of P-gp isoforms by 50-70% and caused a significant 45-65% reduction in the P-gp mediated efflux of R123. Diminished mRNA levels of all three MDR isoforms were seen. LPS-induced AI similarly resulted in significantly reduced levels and activity of P-gp in liver. Although differences in the constitutive levels of P-gp were seen between male and female rats, the influence of AI on P-gp expression and activity was not gender specific. CONCLUSIONS: Experimentally-induced inflammation decreases the in vivo expression and activity of P-gp in liver. This is the first evidence that expression of P-gp is modulated in response to experimentally-induced inflammation.     

Histidase expression is regulated by dietary protein at the pretranslational level in rat liver

Immature Culicoides variipennis (Coquillett) were sampled from aquatic habitats throughout Virginia, reared to adults, and examined by isozyme electrophoresis to assess their taxonomic status. Data from 22 counties showed that C. v. variipennis is widespread and common, the predominant taxon throughout Virginia, and genetically similar to C. v. variipennis in Maryland. Because C. v. variipennis is considered an inefficient vector of the bluetongue viruses, this observation is consistent with the low seroprevalence of bluetongue in indigenous livestock of the mid-Atlantic region. Culicoides v. sonorensis Wirth & Jones, considered to be the primary North American vector of the bluetongue viruses, was recovered in large numbers only from a wastewater lagoon at a dairy in southeastern Virginia, but also was detected at low levels in 6 other counties. Comparison of genetic distances and patterns of discriminating alleles among Virginia populations of C. v. variipennis and C. v. sonorensis showed that respective subspecies are genetically distinct and show no evidence of introgression, irrespective of geographic- and habitat-level sympatry. The persistence of a pure C. v. sonorensis population in a dairy wastewater lagoon may reflect physico-chemical factors that influence the distribution of immature C. variipennis complex populations. A better understanding of the distribution of the C. variipennis complex will benefit regionalization of U.S. exports of livestock and livestock germplasm to bluetongue-free countries.     

Molecular cloning and functional expression of rat liver glutathione-dependent dehydroascorbate reductase

We have isolated a cDNA clone for a novel glutathione-dependent dehydroascorbate reductase from a rat liver cDNA library in lambdagt11 by immunoscreening. The authenticity of the clone was confirmed as follows: first, the antibody that had been purified through affinity for the protein expressed by the cloned lambdagt11 phage recognized only the enzyme in a crude extract from rat liver; and second, two internal amino acid sequences of purified enzyme were identified in the protein sequence predicted from the cDNA. The predicted protein consists of 213 amino acids with a molecular weight of 24,929, which is smaller by approximately 3,000 than the value obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This discrepancy of the molecular weight was explained by post-translational modification because the recombinant protein expressed by a mammalian system (Chinese hamster ovary cells) was of the same size as rat liver enzyme but larger than the protein expressed by a bacterial system (Escherichia coli). Chinese hamster ovary cells, originally devoid of glutathione-dependent dehydroascorbate reductase activity, was made to elicit the enzyme activity (1.5 nmol/min/mg of cytosolic protein) by expression of the recombinant protein. Additionally, the cells expressing the enzyme were found to accumulate 1.7 times as much ascorbate as the parental cells after incubation with dehydroascorbate. This result points to the importance of the dehydroascorbic acid reductase in maintaining a high concentration of ascorbate in the cell.     

Heterogeneous expression of P450 2C12 mRNA in female rat liver

The heterogeneous expression of P450 2C12 mRNA was studied in the rat liver. Liver was sampled from female rat, approximately 40 mg portions being removed from 18 different locations. Total RNA was isolated and subjected to northern blot analysis. The membrane was hybridized with a (32)P-labeled rat P450 2C12 gene-specific oligonucleotide probe. After the hybridized P450 2C12 gene-specific probe had been washed out, the membrane was hybridized again with a rat oligonucleotide probe for the detection of actin mRNA expression. In comparison with actin mRNA expression, P450 2C12 mRNA was not evenly expressed throughout the 18 different locations in the liver. The present results show the heterogeneous expression of P450 2C12 mRNA in the rat liver.