Biomedical subjects

T H Jensen

Publications and source records attributed to T H Jensen.

At least 19 recordsLinked to original sources

PubMed2006

Transcriptome analysis of FSH and FSH variant stimulation in granulosa cells from IVM patients reveals novel regulated genes.

FSH is crucial for oocyte maturation and fertility and is the main component in infertility treatment in assisted reproduction. The granulosa cells expressing the FSH receptor interact with the oocyte and provide nourishing substrates controlling the oocyte maturation. Thus, transcriptome analysis of granulosa cells stimulated by FSH is of major importance in understanding the communication between oocytes and granulosa cells. In this study, gene expression profiles were assessed in human granulosa cells from normal cycling in vitro maturation (IVM) patients using oligonucleotide gene chips. Granulosa cells were stimulated for 2 h with either FSH or a previously generated glycosylated FSH variant (FSH1208) that exhibited increased in vivo activity because of prolonged half-life. The analysis identified 74 significantly FSH/FSH1208 regulated genes. Amongst these were well known FSH regulated genes as well as genes not previously described to be important in the FSH signalling pathway. These novel FSH regulated genes include transcription factors [cAMP responsive element modulator (CREM)/inducible cAMP early repressors (ICER), GATA 6, ZFN 361, Bcl11a, CITED1 and TCF 8] and other regulatory proteins and enzymes (IGF-BP3, syntaxin and PCK1) possibly important for oocyte/granulosa cell interaction and function. Array data were validated for 13 genes by northern blots or RT-PCR. Furthermore, no significant differences in gene regulation were detected between the two FSH analogs. This work uncovers novel data important for understanding the folliculogenesis. Furthermore, the results suggest that FSH1208 has a gene expression profile like FSH and thus, in the light of known prolonged in vivo activity, might be a candidate for improved infertility treatment.

Adolescent↗

PubMed2002

Variations in the severity of phocid herpesvirus type 1 infections with age in grey seals and harbour seals.

The data recorded during an outbreak of phocid herpesvirus type 1 infection among 19 harbour seals and 29 grey seals being nursed in a seal rehabilitation centre in The Netherlands in 1998 were used, together with data from similar outbreaks in previous years, to compare the clinical signs observed in the two species at different ages. The severity of the disease was inversely correlated with age in the harbour seals, and the infected harbour seals generally developed more severe clinical signs than the infected grey seals.

Age Factors↗

PubMed2002

Lactic acidosis in the rectal lumen of patients with septic shock measured by luminal equilibrium dialysis.

BACKGROUND: Gut ischaemia may contribute to morbidity in sepsis, but little is known about the metabolic state of the gut mucosa in such patients. METHODS: Nine patients with abdominal septic shock treated with norepinephrine, and ten healthy subjects, were subjected to equilibrium dialysis with a rectal balloon. pH, PCO(2) and concentrations of L-lactate were measured by auto-analyser. RESULTS: In rectal dialysis fluid from patients with septic shock, acidosis was present (pH 7.23, 95% CI 7.11-7.36) and concentrations of L-lactate were approximately five times greater than controls (2.5-5.8 vs 0.5-1.2 mmol litre(-1)). The lactate concentration was related to the dose of norepinephrine (P<0.001). In contrast, values of dialysate PCO(2) did not differ significantly between patients and controls (6.4-11.0 vs 8.9-13.8 kPa). CONCLUSIONS: The results suggest that, either lactic acidosis in rectal mucosa is related to shock severity, or that norepinephrine causes mucosal ischaemia. In any case, metabolic dysfunction is present in the rectal mucosa in patients with abdominal septic shock treated with norepinephrine.

Acidosis, Lactic↗

PubMed2001

The DECD box putative ATPase Sub2p is an early mRNA export factor.

Nuclear mRNA metabolism relies on the interplay between transcription, processing, and nuclear export. RNA polymerase II transcripts experience major rearrangements within the nucleus, which include alterations in the structure of the mRNA precursors as well as the addition and perhaps even removal of proteins prior to transport across the nuclear membrane. Such mRNP-remodeling steps are thought to require the activity of RNA helicases/ATPases. One such protein, the DECD box RNA-dependent ATPase Sub2p/UAP56, is involved in both early and late steps of spliceosome assembly. Here, we report a more general function of Saccharomyces cerevisiae Sub2p in mRNA nuclear export. We observe a rapid and dramatic nuclear accumulation of poly(A)(+) RNA in strains carrying mutant alleles of sub2. Strikingly, an intronless transcript, HSP104, also accumulates in nuclei, suggesting that Sub2p function is not restricted to splicing events. The HSP104 transcripts are localized in a single nuclear focus that is suggested to be at or near their site of transcription. Intriguingly, Sub2p shows strong genetic and functional interactions with the RNA polymerase II-associated DNA/DNA:RNA helicase Rad3p as well as the nuclear RNA exosome component Rrp6p, which was independently implicated in the retention of mRNAs at transcription sites. Taken together, our data suggest that Sub2p functions at an early step in the mRNA export process.

Active Transport, Cell Nucleus↗

PubMed2001

Quality control of mRNA 3'-end processing is linked to the nuclear exosome.

An emerging theme in messenger RNA metabolism is the coupling of nuclear pre-mRNA processing events, which contributes to mRNA quality control. Most eukaryotic mRNAs acquire a poly(A) tail during 3'-end processing within the nucleus, and this is coupled to efficient export of mRNAs to the cytoplasm. In the yeast Saccharomyces cerevisiae, a common consequence of defective nuclear export of mRNA is the hyperadenylation of nascent transcripts, which are sequestered at or near their sites of transcription. This implies that polyadenylation and nuclear export are coupled in a step that involves the release of mRNA from transcription site foci. Here we demonstrate that transcripts which fail to acquire a poly(A) tail are also retained at or near transcription sites. Surprisingly, this retention mechanism requires the protein Rrp6p and the nuclear exosome, a large complex of exonucleolytic enzymes. In exosome mutants, hypo- as well as hyperadenylated mRNAs are released and translated. These observations suggest that the exosome contributes to a checkpoint that monitors proper 3'-end formation of mRNA.

Cell Nucleus↗

PubMed2001

A block to mRNA nuclear export in S. cerevisiae leads to hyperadenylation of transcripts that accumulate at the site of transcription.

Several factors contribute to nuclear mRNA export in Saccharomyces cerevisiae, including Mex67p, Mtr2p, Gle1p, Nup159p, Dbp5p, and Rip1p. Strains carrying mutations in these factors show rapid and dramatic nuclear accumulation of poly(A)(+) RNA. We have characterized two heat shock mRNAs, SSA4 and HSP104, in these mutant backgrounds; each transcript concentrates in a single intranuclear focus. Evidence suggests that it coincides with the site of transcription. Interestingly, all detectable SSA4 transcripts have undergone 3'-end formation, indicating that RNAs in the foci are no longer nascent. Poly(A) tails of the transcripts are also dramatically longer in all of these export mutants. Based on all of the data, we suggest that very early mRNA maturation events determine transcript export competence.

Active Transport, Cell Nucleus↗

PubMed2001

Long-term results after cemented revision of the femoral component in total hip arthroplasty.

The need for revision total hip arthroplasty has been increasing. The early results have been poor, and different revision techniques have been introduced. We report our results of 84 consecutive cemented first-time revisions of femoral components performed from 1981 through 1988 using a long-stem revision component. The average time to follow-up was 11.4 years (range, 7.9-15.0 years). Patients with 47 revisions had died; 2 of these had been rerevised. Two additional patients were lost to follow-up for other reasons. Of the living patients, 12 had been rerevised, leaving 23 patients (23 hips) for complete follow-up evaluation, including clinical and radiographic assessment. Of 23 patients, 15 reported no pain, 4 had only slight pain, and 4 had more severe pain. In 4 cases, there were definite radiographic signs of loosening of the femoral component. Kaplan-Meier survivorship analysis showed an overall 10-year survival of the femoral component of 77.9%. Using rerevision because of aseptic loosening or definite radiographic loosening as endpoint, the 10-year survival was 80.7%. Simple recementation is well indicated in elderly patients with only minor bone loss.

Aged↗

PubMed2000

[Emergency room patients and alcohol consumption].

To investigate the consumption of alcohol among emergency room patients, we included 395 patients above 18 years old entering the emergency room during a period of ten days. The patients completed a questionnaire about alcohol consumption, smoking habits and medication. In our investigation 56% of the men and 25% of the women had a daily consumption above the limits recommended by the Ministry of Health (three drinks daily for men and two for women), while 41% men and 14% women consumed at least five drinks daily or 35 per week. Trauma seen in male patients with high alcohol consumption was characterised by excessive damage to the head, trauma of the lower limb and blows from an object, person or an animal. The alcoholic women were characterised by excessive chemical injuries, incisions and stab wounds, and trauma of the upper limb. In conclusion a surprisingly large amount of emergency room patients can be defined as alcohol abusers and drinking is found to be associated with excessive damage.

Adult↗

PubMed2000

Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5' RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G(1)-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G(1) but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

Active Transport, Cell Nucleus↗

PubMed1998

The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP.

Nuclear export of intron-containing human immunodeficiency virus type 1 (HIV-1) RNA is mediated by the viral Rev protein that contains both an RNA binding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process that also requires the GTP bound form of the Ran GTPase. Using purified recombinant factors, we show by co-precipitation, gel mobility shift and protein footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leucine residues of the NES and the presence of the cytotoxin leptomycin B (LMB). Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP complex that is sensitive to LMB, NES mutations, and Ran being charged with GTP. Within this complex, CRM1 is readily cross-linked to Cys89 near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and two regions in CRM1 become inaccessible to endoproteinases upon binding suggesting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export receptor for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a preformed Rev-CRM1 complex and specifies a functional interaction with the NES.

Base Sequence↗

PubMed1998

HIV-1 rev nuclear export signal binding peptides isolated by phage display.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is absolutely essential in the viral replication cycle, where it induces the production of viral structural proteins. Rev functions in part by inducing the nuclear export of incompletely spliced mRNA species specified by the presence of an RNA element, the Rev response element (RRE). Several proteins implicated in RNA processing and nucleo-cytoplasmic transport have been shown to interact with Rev, however, their exact roles remain unknown. To map potential protein recognition sites within the Rev structure, we have screened a phage library, displaying random 15-mer peptides, and isolated clones exhibiting similar sequences that specifically interact with Rev. The binding sites on Rev of the corresponding synthetic peptides were characterised by protein footprinting, involving partial proteolysis of radioactively end-labelled Rev protein. Two of the peptides produced a significant footprint within the nuclear export signal of Rev, raising the possibility that they mimic the binding of cellular protein factors implicated in nuclear export.

Amino Acid Sequence↗

PubMed1997

Probing the structure of HIV-1 Rev by protein footprinting of multiple monoclonal antibody-binding sites.

Human immunodeficiency virus type 1 (HIV-1) Rev is a small RNA-binding protein which is essential for viral replication. To investigate the structure of Rev we have mapped the binding sites of a panel of monoclonal antibodies (mAb) by protein footprinting and identified a mAb protecting amino acids within both the N- and C-terminal parts of Rev. Our mapping results support a previously proposed structure (Auer et al., Biochemistry, 33 (1994) 2988-2996) predicting that a helix-loop-helix motif in Rev brings the termini of the protein into proximity. Furthermore, we demonstrate that the binding sites mapped by protein footprinting are in agreement with conventional epitope mapping results and that this technique provides an advantageous strategy for mapping discontinuous sites.

Antibodies, Monoclonal↗

PubMed1996

In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32.

Continuous replication of human immunodeficiency virus type 1 requires the expression of the regulatory protein Rev, which binds to the Rev response element (RRE) and up-regulates the cytoplasmic appearance of singly spliced and unspliced mRNA species. It has been demonstrated that the murine protein YL2 interacts with Rev in vivo and modulates the activity of Rev (Luo, Y., Yu, H., and Peterlin, B. M. (1994) J. Virol. 68, 3850-3856). Here we show that the YL2 human homologue, the p32 protein, which co-purifies with alternative splicing factor ASF/SF2, interacts directly with the basic domain of Rev in vitro and that the Rev-p32 complex is resistant to high concentrations of salt or nonionic detergent. Protein footprinting data suggest that Rev interacts specifically with amino acids within the 196-208 region of p32. An analysis of the ternary complex, formed among p32, Rev, and RRE RNA, shows that Rev can bridge the association of p32 and RRE. Furthermore, we demonstrate that exogenously added p32 specifically relieves the inhibition of splicing in vitro exerted by the basic domain of Rev. Our data are consistent with a model in which p32 functions as a link between Rev and the cellular splicing apparatus.

Amino Acid Sequence↗

PubMed1995

Tools for the production and purification of full-length, N- or C-terminal 32P-labeled protein, applied to HIV-1 Gag and Rev.

We have constructed two new vectors for the production of foreign proteins in Escherichia coli. The vectors, pGEX-GTH and pET-HTG, produce protein fused to glutathione S-transferase (GST) at the N- and C-termini, respectively, allowing one-step purification on glutathione-Sepharose. Furthermore, they carry the recognition sequence (RRASV) for the catalytic subunit of cAMP-dependent heart muscle kinase (HMK) at the terminus distal to the GST tag, enabling specific 32P labeling in vitro. By positioning the GST and HMK sequences at opposite ends of the introduced gene, only full-length fusion protein becomes radiolabeled after purification. Avoiding the labeling of shorter fusion protein species, often observed in bacterial expression of foreign genes, is particularly important for a number of different purposes, including protein mobility shift analysis and protein footprinting technology.

Amino Acid Sequence↗

PubMed1995

Intermolecular binding sites of human immunodeficiency virus type 1 Rev protein determined by protein footprinting.

Human immunodeficiency virus encodes the regulatory protein Rev, which is required for expression of viral structural proteins. It binds to an RNA element (RRE) in the viral transcript and up-regulates the cytoplasmic appearance of unspliced and singly spliced viral mRNA. We have studied the structure of Rev alone and complexed with the RRE and two monoclonal antibodies, using a protein footprinting approach. The method involves radioactive labeling at the C-terminal end of Rev fusion protein followed by limited proteolysis under native conditions, using 10 different proteinases. Rev protein was mainly cleaved within the basic domain and in the C-terminal part. The periodicity of the proteolytic cleavages within the basic domain strongly suggests that it forms an alpha-helical structure with one side facing the solvent. In the presence of RRE, these cleavages became significantly reduced. In addition, strong protection was observed at position 66 outside the basic domain. As a control for the specificity of the footprinting reaction, we confirmed the position of the epitopes for two monoclonal antibodies. This protein footprinting methodology is generally applicable to other proteins for which terminal modifications are acceptable, and provides a useful tool for mapping structure, substrate binding, and conformational changes.

Base Sequence↗

PubMed1995

[Nitrogen oxide inhalation in acute pulmonary failure].

Two cases of adult respiratory distress syndrome (ARDS) treated successfully with nitric oxide (NO) inhalation are described. One patient had severe sepsis and the other had trauma induced ARDS. The slow entry criteria for extracorporeal membrane oxygenation (ECMO) was fulfilled in both cases. NO inhalation substantially improved oxygenation, reduced pulmonary arterial pressure and peak inspiratory pressure. Treatment with NO inhalation was without side effects and easy to administer through the ventilator. Both patients survived without sequelae. We suggest that inhalation with NO should be tried before ECMO treatment is considered in severe ARDS.

Adolescent↗

PubMed1995

Exercise-induced gastric mucosal acidosis.

Gastric acidosis as assessed by tonometry was applied to evaluate changes in splanchnic blood flow during exercise. In six healthy male oarsmen, we determined gastric acidosis in response to 30 min of maximal ergometer rowing. The gastric mucosa carbon dioxide tension was determined by equilibration of isotonic saline to the tonometer. Arterial bicarbonate (HCO3-), pH, arterial oxygen tension (PaO2), and saturation (SaO2) were obtained simultaneously, while pH (pHi) of the gastric mucosa was calculated using the Henderson-Hasselbach equation. During rowing PaO2 and SaO2 decreased to values of 73.7 mm Hg and 95.5%, respectively (P < 0.05). However, during the last minute of rowing the values were normalized with a hyperventilation reducing PaCO2 to 27.1 mm Hg (P < 0.05). Rowing decreased HCO3- from 25.8 (21.4-28.5) to 14.1 (11.6-17.4) mmol l-1, while the gastric carbon dioxide tension increased from 36.8 (24.1-63.9) to 61.7 (48.9-82.0) mm Hg (P < 0.05). Accordingly, pHi decreased from 7.25 (7.04-7.48) to 6.79 (6.67-6.85) (P < 0.05). Arterial pH also decreased (from 7.42 (7.41-7.44) to 7.29 (7.26-7.33) (P < 0.05)), with the enlarged difference between pH and pHi suggesting marked splanchnic hypoperfusion during rowing.

Acidosis↗

PubMed1993

[Translocation and multiple organ failure].

A review of bacterial translocation and multiple organ failure (MOF) is presented. Splanchnic ischaemia plays a central role in the development of MOF, but the exact mechanism of translocation is unclear. The concentration of endotoxins and bacteria in the gut is high. The critically ill patient is often treated with antibiotics with a broad antibacterial spectrum and overgrowth of Gram negative bacteria will take place in the gut favouring the translocation phenomenon. The regime of selective gut decontamination is discussed. Regional and systemic oxygen kinetics together with metabolic markers are important in detecting splanchnic ischaemia. Hepatic vein catheterisation and gastric mucosa pHi are discussed. The regional inflammation in the gut is often initiated by endotoxins, which stimulate the cytokines IL-1, IL-6 and TNF. Another important factor that can accentuate inflammation of the gut is reperfusion injury. A proposal for treatment of splanchnic ischaemia and translocation is discussed i.e.: optimizing central haemodynamic parameters, optimizing the regional microcirculation, treatment with antibodies to endotoxins, gut decontamination and early enteral nutrition. When splanchnic hypoperfusion is detected it cannot be ignored. It may be possible to correct the hypoperfusion with early gastrointestinal resuscitation and to thereby reduce the duration and mortality of MOF. The above mentioned suggestions are all very demanding of resources, but have to be considered in gut directed therapy.

Anti-Bacterial Agents↗