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Biomedical subjects

P Nørgaard

Publications and source records attributed to P Nørgaard.

At least 19 recordsLinked to original sources
PubMed2003

The effect of subclinical hypocalcaemia induced by Na2EDTA on the feed intake and chewing activity of dairy cows.

The effects of induced subclinical hypocalcaemia (SCHC) on feed intake and chewing activity during eating and rumination were studied in dairy cows. Two non-lactating and non-pregnant cows were subjected to three different treatments, with one test per day, such that the plasma free (ionized) calcium (FCa) concentration was maintained at the eucalcaemic level or at one of two constant SCHC levels. The cows and test days followed a 2 x 3 crossover design. SCHC was maintained for 7 h by repeatedly infusing 5% Na2EDTA so that constant FCa concentrations of 0.8 mmol/L or 0.6 mmol/L in plasma were achieved. Control conditions were achieved by infusing isotonic saline. Feed intake and the number of the rumination periods were recorded during test days. The proportion of feed eaten during each test meal (EatPro) was related to the mean plasma FCa. An almost linear decrease in EatPro was observed when the plasma FCa was 0.6-0.9 mmol/L. The cows showed no other clinical signs of hypocalcaemia during Na2EDTA-infusion. The time spent chewing during eating and rumination, and the number of rumination periods during a test day, decreased with a decline in plasma FCa concentration. It was concluded that induced SCHC depresses the feed intake and ruminative activity of dairy cows.

Animals↗
PubMed2001

Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity.

Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.

Amino Acid Motifs↗
PubMed2001

[Uncomplicated gallstone disease and pregnancy].

Pregnancy is associated with an increased incidence of gallstones. However, most gallstones disappear spontaneously after pregnancy without having given any symptoms. New knowledge regarding the pathophysiology confirms changes in the motility of the gallbladder and the flow dynamics and lithogenicity of the bile. The studies have also shown, that these changes resolve completely after labour, which might explain the disappearance of most of the gallstones. These conditions support a conservative attitude towards treatment of gallstone disease during pregnancy. In case cholecystectomy is indicated, laparoscopic intervention seems safe during pregnancy and most optimal during the second trimester where the organogenesis of the fetus is completed and where the size of the uterus does not interfere with the operation technically. Laparoscopic cholecystectomy should under all circumstances be done in departments with routine in laparoscopic operations and with the possibility of gynaecological assistance, in case it is needed.

Cholecystectomy, Laparoscopic↗
PubMed2001

Functional differences in yeast protein disulfide isomerases.

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

Blotting, Western↗
PubMed2000

Salicylate-induced growth arrest is associated with inhibition of p70s6k and down-regulation of c-myc, cyclin D1, cyclin A, and proliferating cell nuclear antigen.

Salicylate and its pro-drug form aspirin are widely used medicinally for their analgesic and anti-inflammatory properties, and more recently for their ability to protect against colon cancer and cardiovascular disease. Despite the wide use of salicylate, the mechanisms underlying its biological activities are largely unknown. Recent reports suggest that salicylate may produce some of its effects by modulating the activities of protein kinases. Since we have previously shown that the farnesyltransferase inhibitor l-744, 832 inhibits cell proliferation and p70(s6k) activity, and salicylate inhibits cell proliferation, we examined whether salicylate affects p70(s6k) activity. We find that salicylate potently inhibits p70(s6k) activation and phosphorylation in a p38 MAPK-independent manner. Interestingly, low salicylate concentrations (</=250 microm) inhibit p70(s6k) activation by phorbol myristate acetate, while higher salicylate concentrations (>/=5 mm) are required to block p70(s6k) activation by epidermal growth factor + insulin-like growth factor-1. These data suggest that salicylate may selectively inhibit p70(s6k) activation in response to specific stimuli. Inhibition of p70(s6k) by salicylate occurs within 5 min, is independent of the phosphatidylinositol 3-kinase pathway, and is associated with dephosphorylation of p70(s6k) on its major rapamycin-sensitive site, Thr(389). A rapamycin-resistant mutant of p70(s6k) is resistant to salicylate-induced Thr(389) dephosphorylation.

Acetaminophen↗
PubMed2000

Optimised extraction of folic acid from multivitamin-mineral preparations for liquid chromatographic analysis.

Degradation of folic acid may occur during extraction of multivitamin-mineral preparations. The degradation may be caused by presence of ions such as Fe3+ and Cu2+, however, the buffer composition may also be critical. This study presents an optimised extraction procedure tested on 24 different products of multivitamin-mineral tablets. The present method yielded mean recoveries of 97% (n = 20) for folic acid and prevented degradation of folic acid in at least 24 h in extracts from multivitamin-mineral tablets.

Chromatography, High Pressure Liquid↗
PubMed2000

Post-exercise facilitation of compound muscle action potentials evoked by transcranial magnetic stimulation in healthy subjects.

Post-exercise facilitation (PEF) of motor evoked potentials (MEPs) was studied by transcranial magnetic stimulation in 15 healthy subjects following standardized and controlled isometric contraction of the biceps brachii muscle. PEF was highly dependent on the time delay (TD) from muscle relaxation to delivery of the magnetic stimulus and only to a minor degree on the duration of the maintained muscular contraction of 2, 4, and 6 s. In addition, PEF was unaffected by the contraction levels of 25%, 50%, and 100% of maximal voluntary contraction (MVC). There was a linear relationship between the log amplitude of the post-exercise MEPs and the TD. The time point at which PEF had vanished was calculated to be 15.2 s. In order to challenge the question whether segmental and/or suprasegmental mechanisms are primarily responsible for PEF, MEPs and H-reflexes were recorded from the soleus muscle following a sustained plantar flexion at the ankle joint in three healthy subjects. PEF of MEPs was present at a TD of 1000 ms following a sustained contraction of 6 s at a level of 50% of MVC. It was accompanied by a pronounced decrease in the soleus H-reflex amplitude at a TD of 1000 ms.

Action Potentials↗
PubMed1999

Inhibition of DNA synthesis by a farnesyltransferase inhibitor involves inhibition of the p70(s6k) pathway.

Previously, the protein farnesyltransferase inhibitor (FTI), L-744, 832, has been shown to inhibit the proliferation of a number of tumor cell lines in vitro in a manner that correlated with the inhibition of the mitogen-activated protein kinase cascade. Here we show that FTI inhibits p70(s6k) phosphorylation in mammary tumors in vivo in transgenic mice. Furthermore, in a mouse keratinocyte cell line, FTI inhibits p70(s6k) phosphorylation and activity and inhibits PHAS-1 phosphorylation in vitro in both rapidly growing cells and in growth factor-stimulated quiescent cells. Dominant-negative Ras expression inhibits p70(s6k) stimulation by epidermal growth factor, demonstrating a requirement for Ras activity during p70(s6k) activation. FTI does not inhibit protein kinase B phosphorylation on Ser473, indicating that FTI does not act by inhibiting phosphatidylinositol 3-kinase. FTI also inhibits DNA synthesis in keratinocytes, and inhibition of DNA synthesis correlates closely with p70(s6k) inhibition. Rapamycin, an inhibitor of p70(s6k) and PHAS-1 phosphorylation, causes a 30-45% reduction in DNA synthesis in keratinocytes, while FTI induces an 80-90% reduction in DNA synthesis. These observations suggest that alteration of p70(s6k) and PHAS-1 function by FTI are responsible for a substantial portion of the growth-inhibitory properties of FTI. Together, these data demonstrate that p70(s6k) and PHAS-1 are novel downstream targets of FTI and suggest that the anti-tumor properties of FTI are probably due to the inhibition of multiple mitogenic pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines.

Transforming growth factor beta (TGF-beta) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-beta due to lack of type II receptor (RII) has been described in some cancer types including small cell lung cancer (SCLC). The purpose of this study was to examine the cause of absent RII expression in SCLC cell lines. Northern blot analysis showed that RII RNA expression was very weak in 16 of 21 cell lines. To investigate if the absence of RII transcript was due to mutations, we screened the poly-A tract for mutations, but no mutations were detected. Additional screening for mutations of the RII gene revealed a GG to TT base substitution in one cell line, which did not express RII. This mutation generates a stop codon resulting in predicted synthesis of a truncated RII of 219 amino acids. The nature of the mutation, which has not previously been observed in RII, has been linked to exposure to benzo[a]-pyrene, a component of cigarette smoke. Since RII has been mapped to chromosome 3p22 and nearby loci are often hypermethylated in SCLC, it was examined whether the lack of RII expression was due to hypermethylation. Southern blot analysis of the RII promoter did not show altered methylation patterns. The restriction endonuclease pattern of the RII gene was altered in two SCLC cell lines when digested with Smal. However, treatment with 5-aza-2'-deoxycytidine did not induce expression of RII mRNA. Our results indicate that in SCLC lack of RII mRNA is not commonly due to mutations and inactivation of RII transcription was not due to hypermethylation of the RII promoter or gene. Thus, these data show that in most cases of the SCLC cell lines, the RII gene and promoter is intact in spite of absent RII expression. However, the nature of the mutation found could suggest that it was caused by cigarette smoking.

Blotting, Northern↗
PubMed1999

Treatment with farnesyl-protein transferase inhibitor induces regression of mammary tumors in transforming growth factor (TGF) alpha and TGF alpha/neu transgenic mice by inhibition of mitogenic activity and induction of apoptosis.

Mouse mammary tumor virus-transforming growth factor alpha (MMTV-TGF alpha) and MMTV-TGF alpha/neu transgenic mice develop mammary tumors after a long latency and therefore provide useful model systems for breast cancer with its recognized activation of receptor tyrosine kinase signaling. We used these mice to study the antitumor effect of L-744,832 (FTI), a potent and selective inhibitor of farnesyl-protein transferase, and hence of Ras function. A total of 55 mice were assigned randomly to treatment with FTI or vehicle, and one-half of the mice were crossed over after initial treatment to the opposite group. L-744,832 induced reversible regression of mammary tumors that was paralleled by a decrease in serum levels of TGF alpha secreted by the tumor cells. There was no difference in response to treatment with FTI between MMTV-TGF alpha mice, in which tumorigenesis was accelerated by multiparity or the chemical carcinogen 7,12-dimethylbenzanthracene, and MMTV-TGF alpha/neu mice. The tumor histological type had no impact on FTI sensitivity. For mechanistic analyses, tumor excision biopsies were obtained from 12 mice before and after treatment with L-744,832. In these samples, tumor regression was paralleled biochemically by inhibition of mitogen-activated protein kinase activity and biologically by an increase in G1-phase and decrease in S-phase fractions, as well as induction of apoptosis. These results suggest that the potential clinical use of FTI could be expanded to include cancers harboring activated receptor tyrosine kinases as well as those containing activated Ras.

Alkyl and Aryl Transferases↗
PubMed1998

High value of the radiobiological parameter Dq correlates to expression of the transforming growth factor beta type II receptor in a panel of small cell lung cancer cell lines.

Our panel of SCLC cell lines have previously been examined for their radiobiological characteristics and sensitivity to treatment with TGF beta 1. In this study we examined the possible correlations between radiobiological parameters and the expression of the TGF beta type II receptor (TGF beta-rII). We have, in other studies, shown that the presence of TGF beta-rII was mandatory for transmitting the growth inhibitory effect of TGF beta. The results showed a statistically significant difference in Dq, i.e. the shoulder width of the survival curve, between cell lines expressing TGF beta-rII and cell lines which did not express the receptor (P = 0.01). Cell lines expressing TGF beta-rII had a high Dq-value. TGF beta-rII expression did not correlate with any other radiobiological parameters. We suggest that an intact growth inhibitory pathway mediated by the TGF beta-rII may have a significant role for the repair of radiation induced DNA damage in SCLC.

Blotting, Northern↗
PubMed1998

Fibrolytic enzyme treatment of barley grain and source of forage in high-grain diets fed to growing cattle.

We conducted a study to determine the effects of treating barley grain with a fibrolytic enzyme mixture on chewing activities, ruminal fermentation, and total tract digestibility in cattle. We also investigated the potential benefits of using barley straw rather than barley silage as a roughage source in high-grain diets for feedlot cattle. Steers were given ad libitum access to one of four diets that consisted of 95% barley-based concentrate and 5% forage (DM basis). The concentrate was either control or enzyme-treated, and the forage was either barley silage or barley straw. Applying the enzyme mixture onto the barley lowered the concentrations of dietary ADF and NDF. However, it is not certain when this fiber hydrolysis occurred relative to feed consumption because the fiber analyses were conducted after the study was completed. Enzyme treatment of barley increased total tract dietary ADF digestibility by 28% (P<.05). Acetate-to-propionate ratio tended to decrease, which suggests that enzymes may have increased ruminal starch digestion as a result of enhanced digestion of barley hulls. Replacing silage with straw increased ADF intake (P<.05) and resulted in 1-h/d increase in rumination time (P<.05). Even though there was no effect of diet on ruminal pH, replacing silage with straw increased ruminal acetate, as a percentage of total VFA, and total tract ADF digestion (P<.01). This study demonstrates that using a fibrolytic enzyme mixture in high-grain diets that contain mainly barley grain can improve fiber digestion and grain utilization, but the mode of action is unclear. Straw can be used rather than silage to increase the effective fiber content of a high-grain feedlot diet.

Amylases↗
PubMed1996

Acquired TGF beta 1 sensitivity and TGF beta 1 expression in cell lines established from a single small cell lung cancer patient during clinical progression.

Three small cell lung cancer cell lines established from a single patient during longitudinal follow-up were examined for in vitro expression of TGF beta and TGF beta receptors, i.e. the components of an autocrine loop. GLC 14 was established prior to treatment, GLC 16 on relapse after chemotherapy and GLC 19 on recurrence after radiotherapy. TGF beta was detected by ELISA and TGF beta receptors by chemical crosslinking to radiolabelled TGF beta 1. Furthermore, TGF beta and TGF beta receptor mRNAs were detected by northern blot analysis. Expression of type II TGF beta receptor mRNA and protein was found in GLC 16 and GLC 19. These cell lines were also growth inhibited by exogenously administrated TGF beta 1. TGF beta 1 mRNA and protein in its latent form was only expressed in the radiotherapy-resistant cell line, GLC 19. The results indicate that disease progression in this patient was paralleled by a gain in sensitivity to the growth inhibition by TGF beta 1 due to type II TGF beta receptor, and a gain of latent TGF beta 1 protein. Lack of type II receptor expression in GLC 14, which was also resistant to growth inhibition by exogenous TGF beta 1, was not due to gross structural changes in the type II receptor gene, as examined by Southern blotting. Also, the type I receptor could not be detected by ligand binding assay in this cell line, despite expression of mRNA for this receptor. This agrees with previous findings that type I receptor cannot bind TGF beta 1 without co-expression of the type II receptor.

Blotting, Northern↗
PubMed1996

Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines.

In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co-expression of beta-glycan, RI and RII.

Activin Receptors, Type I↗
PubMed1996

Technical note: reversible re-entrant cannulation of the parotid duct in cattle using a new injection anesthesia regimen.

A new method for reversible reentrant cannulation of the parotid duct in cattle by use of an injectable anesthetic regimen is described. Ruminal contents were removed before anesthesia instead of food deprivation. The parotid duct was dilated by means of a long hemostatic forceps: its jaws were encased in a piece of plastic tube, and the tip was extended by a piece of flexible wire. A polyethylene parotid catheter was inserted into the dilated duct and passed through the cheek. The saliva was re-directed back into the mouth via a silicone tube through another fistula in the cheek. The cannulations were maintained for 3 to 11 wk and two cows were recannulated three to five times in both parotid ducts without problems. No pathological changes of the mouth epithelium were found at slaughter. The rate of flow from the ducts during rest, ruminating, and eating were 5 to 20, 25 to 50, and 40 to 75 mL/min, respectively.

Anesthesia, Intravenous↗
PubMed1994

Growth suppression by transforming growth factor beta 1 of human small-cell lung cancer cell lines is associated with expression of the type II receptor.

Nine human small-cell lung cancer cell lines were treated with transforming growth factor beta 1 (TGF-beta 1). Seven of the cell lines expressed receptors for transforming growth factor beta (TGF-beta-r) in different combinations between the three human subtypes I, II and III, and two were receptor negative. Growth suppression was induced by TGF-beta 1 exclusively in the five cell lines expressing the type II receptor. For the first time growth suppression by TGF-beta 1 of a cell line expressing the type II receptor without coexpression of the type I receptor is reported. No effect on growth was observed in two cell lines expressing only type III receptor and in TGF-beta-r negative cell lines. In two cell lines expressing all three receptor types, growth suppression was accompanied by morphological changes. To evaluate the possible involvement of the retinoblastoma protein (pRb) in mediating the growth-suppressive effect of TGF-beta 1, the expression of functional pRb, as characterised by nuclear localisation, was examined by immunocytochemistry. Nuclear association of pRb was only seen in two of the five TGF-beta 1-responsive cell lines. These results indicate that in SCLC pRb is not required for mediation of TGF-beta 1-induced growth suppression.

Carcinoma, Small Cell↗