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H Jacobsen

Publications and source records attributed to H Jacobsen.

At least 37 records · Page 2Linked to original sources
PubMed1998

Caspase-mediated cleavage is not required for the activity of presenilins in amyloidogenesis and NOTCH signaling.

The Alzheimer's disease (AD) associated presenilin (PS) proteins are proteolytically processed. One of the processing pathways involves cleavage by caspases. Pharmacological inhibition of caspases is currently being discussed as a treatment for a variety of neurodegenerative diseases, including AD. We therefore inhibited caspase mediated processing of PS-1 and PS-2 in cells transfected with wt and mutant PS by mutagenizing the substrate recognition site or by using specific peptide aldehydes known to block caspases. We found that the inhibition of caspase mediated processing of PS proteins does not decrease its amyloidogenic activity. PS cDNA constructs with mutations in the caspase cleavage site are biologically active in Caenorhabditis elegans such as the wt human PS proteins, demonstrating that caspase-mediated cleavage is not required for the physiological PS function in NOTCH signaling.

Alzheimer Disease↗
PubMed1998

Alzheimer's disease associated presenilin-1 holoprotein and its 18-20 kDa C-terminal fragment are death substrates for proteases of the caspase family.

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease leading to the formation of two stable fragments of approximately 30 and approximately 20 kDa [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. In addition to the conventional fragments, alternative cleavage products were observed as well. Here we characterize an alternative proteolytic pathway of PS-1 which involves proteases of the caspase superfamily. Caspase mediated cleavage occurs between aspartate 345 and serine 346 C-terminal to the conventional cleavage determined previously [Podlisny, M., et al., (1997) Neurobiol. Dis. 3, 325-337]. Full-length PS-1 can serve as a substrate for caspase-like proteases, as demonstrated by the generation of the alternative C-terminal fragment in cells expressing PS-1 containing the Deltaexon 10 deletion which is known to accumulate as a full-length molecule [Thinakaran, G., et al. (1996)]. By inhibition of de novo protein synthesis in untransfected cells we demonstrate that the conventional C-terminal fragment of PS-1 is a substrate for caspase-like proteases as well. Therefore full-length and the conventional C-terminal fragment of PS-1 can serve as potential death substrates. Due to the fact that very little full-length PS-1 is expressed in vivo, the much more abundant C-terminal fragment and not the full-length precursor is the major in vivo substrate for the alternative cleavage of PS-1 by proteases of the caspase superfamily.

Alzheimer Disease↗
PubMed1998

Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos.

Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000 degrees C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over -180 degrees C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty-one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction.

Animals↗
PubMed1998

Eating pattern and self-esteem in overweight women.

Recent studies have shown that the relationship between poor self-esteem and disturbed eating patterns may be more fully understood when the self-esteem concept is divided into an affective domain ('self-liking') and a cognitive domain ('self-competence'). In the present study 38 overweight women between the ages of 21 and 68 participated in an 8-week weight reduction program whereby the Self-Liking and Competence Scale [SLCS] and the Eating Disorder Scale [EDS-5] were used in order to measure self-esteem and eating patterns, respectively. An improvement in the eating patterns corresponded to an improvement in self-liking, but not in self-competence. Statistically significant weight reduction did occur, but on the average, the subjects still remained overweight. As in other studies on normal weight individuals in analogous test situations completing the same instruments, the results point only to a specific relationship between disturbed eating patterns and self-liking. The results suggest that an increased benefit from a weight reduction program could be expected if strategies for improving eating patterns and self-liking are included. This hypothesis warrants further controlled treatment studies.

Adult↗
PubMed1998

Comparison of six sets of PCR primers from two different genomic regions for amplification of GB virus C/hepatitis G virus RNA.

Forty-four clinical samples positive for GB virus C (GBV-C)/hepatitis G virus (HGV) were tested with six primer sets, four from the 5' untranslated region (5'-UTR) and two from the NS5a genomic region. Two of the 5'-UTR primer sets, when used in a single-round 60-cycle PCR, detected between 86.4 and 97.7% of the positive samples, while two different sets from the same area, when used in a nested PCR, amplified between 97.7 and 100% of the positive specimens. Both sets from the NS5a region, when used in a single-round PCR, detected 95.5% of the GBV-C/HGV-positive samples. Parallel testing with two PCR sets, one from the 5'-UTR and a second from NS5a, may eliminate false-negative results attributable to the genetic heterogeneity of the virus.

DNA Primers↗
PubMed1997

Electrically induced deposition of metal on the human skin.

The diagnosis of electrical lesions may be difficult. Forensic material consisting of 24 post-mortem skin samples from 11 fatalities due to electricity was examined with regard to the deposition of iron or copper on the skin. Metal deposition was demonstrated on the epidermis in eight cases. It is concluded that staining for iron and copper may improve the diagnostic sensitivity of electrical lesions.

Adolescent↗
PubMed1997

Presenilins are processed by caspase-type proteases.

Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolyzed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed.

Amino Acid Sequence↗
PubMed1997

[Sjögren's syndrome. New diagnostic aspects].

European criteria for classification of Sjögren's syndrome have recently been developed and evaluated. We report the clinical and laboratory findings in 96 patients with primary Sjögren's syndrome who have been classified according to these new criteria. In our patient population the latency from appearance of the first symptom to diagnosis was 11 years. In addition to sicca symptoms in mucous membranes, the dominant symptoms were periodic fatigue (92%), arthralgia (82%), hoarse voice (71%), dry cough (54%) and diarrhoea (51%). Antibodies to the nuclear antigens SSA and SSB were found in respectively 22.2% and 15.6% of the patients. Two out of ten patients with both anti-SSA and anti-SSB antibodies gave birth to a child with heart block.

Autoantibodies↗
PubMed1997

Evaluation of hepatitis C virus envelope proteins expressed in E. coli and insect cells for use as tools for antibody screening.

BACKGROUND/METHODS: The two envelope proteins of hepatitis C virus, E1 and E2, were expressed in E. coli and, as secretory proteins, in Sf9 insect cells using recombinant baculoviruses. Co-infection of insect cells with E1 and E2-recombinant baculoviruses was performed, which has been shown to result in formation of E1-E2 dimers. All envelope proteins were purified by Ni2+-NTA chromatography and used for screening of serum samples in a HCV EIA assay. Serum samples of normal blood donors, chronically HCV-infected patients, a mixed titer panel and several seroconversion panels were screened and compared to test results with Cobas Core Anti-HCV EIA. RESULTS: Screening of the sera of chronically HCV-infected patients (100% positive in Cobas Core Anti-HCV EIA) revealed 10-40% anti-E1 positive sera using different Sf9-expressed, glycosylated proteins and 93% using E. coli-expressed, non-glycosylated E1 protein. When the same sera were tested with different E2 proteins expressed in Sf9 cells and in E. coli, about 70-73% showed anti-E2 reactivity. When the proteins from Sf9 cells co-infected with E1- and E2-recombinant baculoviruses were tested, 70-80% of the same sera showed anti-envelope reactivity. CONCLUSIONS: Testing of these patient antisera, and those from the well-characterized mixed titer panel BBI-PHV203, showed that recombinant E1 expressed in E. coli and co-expressed E1 and E2 proteins from Sf9 cells could be used as additional tools for anti-HCV antibody screening.

Animals↗
PubMed1997

Emergence of resistant variants of HIV in vivo during monotherapy with the proteinase inhibitor saquinavir.

We examined the phenotypic and genotypic properties of virus from the peripheral blood mononuclear cells (PBMC) and plasma of eight HIV-1-infected asymptomatic patients before and during monotherapy with the proteinase inhibitor saquinavir. Susceptibility of primary isolates to drug was assessed in PBMC culture by deriving IC50 and IC90 values. The observed increases in IC50 and IC90 after approximately one year of therapy with a dosage of 600 mg tds suggests the presence of virus resistant to saquinavir in vivo. The magnitude of this altered susceptibility ranged from three-fold to in one case 100-fold. In two patients a greater than eight-fold decrease in susceptibility to saquinavir was observed. Sequencing of the proteinase genes in viral RNA obtained from patient plasma and/or PBMC was carried out by PCR in parallel with sensitivity testing. In each case between nine and 12 clones were analysed. In the two patients from whom virus had greater than eight-fold reduction in susceptibility, a point mutation was observed in the viral proteinase (Leu90--> Met/Ile). Further mutations were observed at residues 36, 71 and 84 in these subjects. In a third patient, in whom an eight-fold increase in HIV IC50 of saquinavir was observed, no mutations were detected in the proteinase; sequencing of proteinase cleavage sites in viral gag-pol revealed no significant mutations. In no patient was a Gly48-->Val mutation observed, although this has been associated with resistance in vitro. The Leu90-->Met mutation was observed in five subjects, but a greater than eight-fold phenotypic change in antiviral susceptibility was seen in only two of these. Hence, in vivo, the Leu90-->Met but not the Gly48-->Val mutation is necessary, but not sufficient, for phenotypic resistance to saquinavir in HIV.

Adult↗
PubMed1997

Ten days of head down tilt: effects of isotonic and hypertonic saline loads in normal man.

The initial response to bed rest involves an increase in central blood volume leading to a an enhanced renal excretion of fluid and electrolytes. Within 24 hours of head-down bed rest a new steady state condition occurs with a sustained reduction of plasma volume, extracellular fluid volume, total body water, and body weight. It was the purpose of the present study to elucidate the volume homeostatic mechanisms during head-down bed rest by investigating the endocrine and renal responses to a load of sodium chloride given as either an isotonic or a hypertonic solution.

Angiotensin II↗
PubMed1997

Multiple sclerosis in the Faroe Islands. 7. Results of a case control questionnaire with multiple controls.

Detailed questionnaires were completed in 1978-79 by 23 of the 28 then known resident Faroese multiple sclerosis (MS) patients and 127 controls. These controls were divided into 69 Group A (patient sibs and other relatives), 37 Group B (matched neighbor controls, their spouses and sibs, plus patient's spouse), and 21 Group C (distant matched controls, spouses, relatives living where MS patients never resided and British troops were not encamped during the war). No differences between cases and controls were found for education, occupation, types of residence, bathing, sanitary or drinking facilities, and nature of house construction or heating. Detailed dietary histories, available for half the subjects, revealed no differences, cases versus controls, for four age periods between age 0 and 30 years, and for 16 specified foodstuffs. Animal exposures showed overall no consistent differences by location or type of animal. There was a tendency to greater exposure to British troops during the war for cases versus Groups A and B but this did not attain statistical significance. Vaccinations for smallpox, tetanus and diphtheria were less common in the MS; no difference was found for other vaccinations. Except for a relative deficit in the cases for rubella and (insignificantly) for measles, mumps and chicken pox, reported illnesses were equally common among all groups. Operations, hospitalizations and injuries did not differentiate the groups, nor did age at menarche for women. Neurologic symptoms were significantly more common in the cases than in the controls.

Adolescent↗
PubMed1996

Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase.

The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine P19 core with NI(2+)-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. Lyko et al. (1995) J. Biol. Chem. 270, 19873-19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein.

Amino Acid Sequence↗
PubMed1996

Tumor necrosis factor receptor p55 mediates induction of HIV type 1 expression in chronically infected U1 cells.

Tumor necrosis factor alpha (TNF-alpha) is a potent inducer of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected cells. The aim of this study was to investigate the role played by the two known TNF-alpha receptors, TNFR-p55 and TNFR-p75, in the activation of HIV-1 expression. As a model system the latently infected human promonocytic cell line U1 was stimulated with wild-type TNF-alpha, with TNF-alpha muteins that specifically bind to one or the other receptor or with receptor-specific monoclonal antibodies. Induction of HIV-1 expression, measured by p24 core antigen capture enzyme-linked immunosorbent assay (ELISA), was found to be exclusively triggered by TNFR-p55 stimulation. However, our results also showed that the addition of TNFR-p75-specific ligands negatively modulated the HIV-1 expression induced via TNFR-p55.

Antigens, CD↗
PubMed1996

Reduced sensitivity to saquinavir: an update on genotyping from phase I/II trials.

A genotypic analysis of the HIV-1 proteinase was performed on clinical specimen obtained from patients after different periods of Saquinavir (SQV) treatment. Proteinase genes of integrated proviral DNA from PBMC were isolated by PCR, cloned and individual sequences were obtained. Genotypic resistance was defined by the Gly48-->Val and Leu90-->Met exchanges. Frequencies and kinetics of resistance development will be reported for phase I/II trials V13330. V13329, O13328 and ACTG229 in patients on monotherapy or combination therapy with RT inhibitors. Data from V13330 have been analysed in more detail for correspondence of genotypic and phenotypic resistance and any correlation between resistance and changes in plasma viral RNA load. Furthermore, we will discuss the data from our extensive proteinase gene sequence collection with respect to mutational changes which would be indicative of resistance to other inhibitors of HIV-1 proteinase.

Drug Resistance, Microbial↗
PubMed1996

Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells.

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.

Adenosine Triphosphate↗