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Biomedical subjects

M Horst

Publications and source records attributed to M Horst.

At least 55 records · Page 3Linked to original sources
PubMed1993

Desalting electroeluted proteins with hydrophilic interaction chromatography.

We describe a chromatographic procedure for the removal of sodium dodecyl sulfate (SDS) from proteins isolated by electroelution. It involves chromatography of electroeluates on poly(2-hydroxyethyl-aspartamide) silica, a support initially developed for hydrophilic interaction chromatography. The electroeluate, dialyzed against ammonium bicarbonate-SDS buffer, is directly injected onto the column, which is equilibrated in an n-propanol concentration greater than 60%. Bound proteins are eluted with a gradient of decreasing n-propanol. This procedure removes essentially all of the Coomassie blue-related contaminants and separates SDS from the protein. Due to the use of volatile buffer systems, the proteins are recovered in completely salt-free form, which facilitates further protein manipulation. After removal of the organic solvent from the chromatographic desalting step, the recovered proteins are directly amenable to N-terminal protein sequencing and, after evaporation of the organic phase, to enzymatic digestions and subsequent separation of fragments by reverse-phase HPLC.

Amino Acid Sequence↗
PubMed1992

Identification of a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane.

Import of proteins into mitochondria involves the cooperation of protein translocation systems in the outer and inner membranes. We have identified a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane. This 45-kDa protein could be crosslinked to a partly translocated precursor, which cannot be imported across the inner membrane when the matrix is depleted of ATP. In addition, an antibody against this protein strongly inhibited protein import into right-side-out inner-membrane vesicles. The 45-kDa protein accounts for only 0.1% of mitochondrial protein and appears peripherally attached to the outer face of the inner membrane. The properties of this protein suggest that it is a component of the protein import system of the mitochondrial inner membrane.

Biological Transport↗
PubMed1991

Biosynthesis of glycosylated human lysozyme mutants.

Complementary DNA encoding human lysozyme was subjected to oligonucleotide-directed mutagenesis. At one of three selected positions, amino acid residues 22, 68, or 118, the signal for N-linked glycosylation was created. The mutant DNAs were inserted into a eucaryotic vector and transfected into cultured hamster cells. The three mutant cDNAs directed synthesis of lysozyme mutants, which were named LI, LII, and LIII. The mutant lysozymes LI and LII comprised mixtures of glycosylated and nonglycosylated forms. The glycosylated and nonglycosylated forms of mutant LI were found to have an enzymatic activity similar to normal human milk lysozyme. The usage of the glycosylation sites in the mutants was similar in Chinese hamster ovary (CHO) and baby hamster kidney cells. Approximately two of every three molecules in mutant LI, approximately one of every eight molecules in mutant LII, and practically no molecules in mutant LIII became glycosylated. In CHO cells, the processing of the oligosaccharide side chains yielded several larger products than in baby hamster kidney cells. This size variability of glycosylated lysozyme from CHO cells may be explained by the presence of biantennary and triantennary endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides with N-acetyllactosamine repeats of variable length and by the presence of hybrid oligosaccharides, as suggested by affinity to several lectins and sensitivity to endo-beta-galactosidase. In both cell types, the majority of the glycosylated forms were secreted and thus behaved similarly to nonglycosylated lysozyme. A small proportion of mutant LI lysozyme remained associated with the cells. The retained lysozyme was recruited predominantly from the molecules bearing high mannose oligosaccharides. These molecules were targeted to lysosomes, and their carbohydrate was trimmed to an endo-beta-N-acetylglucosaminidase H-resistant form. Owing to the small size of mutant LI lysozyme, minor changes in the size of its carbohydrate moiety result in detectable changes in the electrophoretic mobility of the whole glycoprotein. We suggest that this novel glycoprotein could be used as a reporter in studies on processing and segregation of glycoproteins.

Animals↗
PubMed1991

Expression and maturation of human cathepsin D in baby-hamster kidney cells.

In medium and in homogenates from baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA, an elevated activity of cathepsin D was found as compared to non-transfected cells. The elevated activity was removed by titrating the homogenates with an anti-(human cathepsin D) antibody. Metabolic labelling and immunoprecipitation revealed that, in the transfected cells, human cathepsin D was synthesized as a 53-kDa precursor indistinguishable from that found in human cells. A portion of the precursor was secreted and the remainder was processed to intermediate and mature chains within a few hours of synthesis. The precursor that was released from the transfected cells had a slightly smaller apparent size than that from cultured human fibroblasts. This difference was abrogated when the precursors were treated with glycopeptidase F. In the intracellular small chain a difference was observed in the size of carbohydrate chains that were cleavable with endo-beta-N-acetylglucosaminidase H. Sequence analysis of the N-termini of mature intracellular cathepsin D indicated a N-terminal trimming in both large and small chains from both human and transfected hamster cells. The proteolytic maturation of human cathepsin D in BHK cells closely resembles that in human cells, whereas a portion of the carbohydrate side chains is processed differently. The trimming of the N-termini in mature cathepsin D is proposed to be a part of the maturation and aging of this protein.

Acetylglucosaminidase↗
PubMed1991

Differential segregation of human and hamster cathepsin D in transfected baby-hamster kidney cells.

The segregation of human cathepsin D, studied in baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA and compared with that of hamster cathepsin D in the same cells, showed that, in cells that expressed human cathepsin D at a low rate, most of the enzyme remained intracellular. In contrast, when the enzyme was expressed at a high rate, most was secreted. The segregation was examined with an anti-(human cathepsin D) antibody that reacted with the human enzyme exclusively and an anti-(rat cathepsin D) antibody that reacted with both enzymes. In one protocol the cells were metabolically labelled and the two antibodies were used in sequence to precipitate the enzymes from extracts of cells and medium. High expression of the human enzyme did not interfere with the segregation of hamster cathepsin D. In another protocol the activity of cathepsin D in cells and medium was measured before and after titration with anti-(human cathepsin D) antiserum. Human cathepsin D was found predominantly in the medium, and hamster cathepsin D mainly in the cells. In the presence of 10 mM-NH4Cl the intracellular segregation of hamster cathepsin D was strongly inhibited, while the segregation of human cathepsin D was only slightly diminished. In BHK cells, at least two systems participate in the sorting of the two cathepsins, one of them being rather insensitive to NH4Cl.

Ammonium Chloride↗
PubMed1990

Double-blind, placebo-controlled rush immunotherapy with a standardized Alternaria extract.

Specific immunotherapy is ineffective with unstandardized mold extracts. A double-blind, placebo-controlled study was performed in 24 patients (5 to 56 years of age) only allergic to Alternaria. The extract was standardized by isoelectric focusing, crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, RAST inhibition, and skin tests and contained allergen Alternaria major allergen a I and antigen B. Thirteen patients received the active treatment, and 11 received the placebo. Immunotherapy was started by a 2-day rush protocol; maintenance injections were administered for 1 year. The patient's self-evaluation of the treatment was significantly (p less than 0.001) lower in the placebo-treated group. Global symptom-medication scores, including asthma and rhinoconjunctivitis, were significantly (p less than 0.005) lower in the actively treated group. Nasal challenges with Alternaria extract were performed before immunotherapy and after 1 year of treatment. There was no difference in the placebo-treated group and a significantly (p less than 0.01) increased mean provocative dose in the actively treated group. Skin tests were significantly reduced in the actively treated group. Specific IgG increased significantly in the actively treated group and were stable in the placebo-treated group. There was a significant correlation between nasal challenges and nasal symptom-medication scores (p less than 0.03) or the patient's self-evaluation of efficacy (p less than 0.05). This study demonstrated that patients only sensitized to Alternaria benefit from specific immunotherapy with a standardized Alternaria extract.

Adolescent↗
PubMed1990

Symptoms, diagnosis, and therapy of primary sarcomas of the pulmonary artery.

Primary sarcoma of the pulmonary artery is a rare tumor which must be considered in differential diagnosis of pulmonary embolism. The data of the 93 cases published up to now is evaluated synoptically after a report on a primary sarcoma of the pulmonary artery. Modern diagnostic imaging methods (pulmonary DSA, perfusion scintigraphy, echocardiography, computer tomography, nuclear magnetic resonance tomography) may corroborate the suspicion of a sarcoma of the pulmonary artery. With a median survival time of 1.5 months, the prognosis of patients with primary sarcomas of the pulmonary artery must be regarded extremely poor. After tumor excision, the median survival time of the patients could be prolonged (statistically significant; p less than 0.01) to ten months. The significance of adjuvant chemotherapy and/or radiotherapy cannot be appraised at present.

Diagnosis, Differential↗
PubMed1989

Calcitriol enhances transcriptional activity of lysozyme and cathepsin D genes in U937 promonocytes.

Calcitriol (1,25-dihydroxyvitamin D3) has been reported to enhance the rate of synthesis of lysozyme [Rouis, Thomopoulos, Louache, Testa, Heroy & Titeux, (1985) Exp. Cell Res. 157, 539-543] and of cathepsin D [Stein, Braulke, von Figura & Hasilik (1987) Biol. Chem. Hoppe-Seyler 368, 413-418] in human promonocytes U937. In this study we show hat the hormone enhances the steady-state levels of cathepsin D and lysozyme mRNAs. The enhancement of the latter but not of the former is strongly inhibited by cycloheximide. The degradation rates of cathepsin D and lysozyme mRNAs as revealed in the presence of actinomycin D are not significantly affected by the treatment of the cells with calcitriol. In nuclei prepared from calcitriol-treated cells, an enhanced rate of synthesis of cathepsin D and lysozyme mRNA precursors is observed. These results suggest that in human promonocytes the rate of the transcription of cathepsin D and lysozyme genes is enhanced in the presence of calcitriol and that the regulation by the hormone of the transcription of lysozyme gene involves a short-lived or an induced protein.

Calcitriol↗
PubMed1988

Comparison between modified skin prick test with standardized allergen extracts and Phazet.

Skin prick tests (SPTs) represent one of the most common techniques of skin testing and are of great value, especially when standardized extracts are used. Phazet is a puncture test involving needles coated with standardized allergens. Phazet and SPTs have been compared in 130 subjects tested with six standardized allergen extracts: orchard-grass, olive, and mugwort pollens, cat dander, Dermatophagoides pteronyssinus, and Alternaria. Patients have been carefully selected on the basis of a suggestive clinical history, SPTs, and RAST. For all allergenic species, at least 20 nonsensitive individuals have been tested. The sensitivity (true positive in percent of patients with disease) of Phazet was 99.4%, and its specificity (true negative in percent of patients without disease) was 99.2%. It is concluded that Phazet and SPTs with standardized extracts are equally effective in the diagnosis of immediate-type allergy and that standardized extracts have improved the diagnosis of allergy, especially for previously poorly characterized extracts, such as Alternaria.

Adolescent↗
PubMed1985

The influence of vertebral body fracture, intradiscal injection, and partial discectomy on the radial bulge and height of human lumbar discs.

The effects of vertebral body fracture, intradiscal injection, and partial discectomy on the radial extension and height of human lumbar discs have been investigated in vitro. Fracture and discectomy result in an increase of the radial disc bulge and a decrease of the disc height; intradiscal injection results in a decrease of the radial disc bulge and in an increase of the disc height. The significance of these changes is assessed by comparing them with those changes induced by load decrease or increase within the physiologic range. The results may explain clinical observations with regard to the persistence of or relief from low-back pain after fractures or after application of the therapeutic measures simulated in this experiment.

Cadaver↗
PubMed1984

Inaccuracy of diagnostic peritoneal lavage in penetrating colonic trauma.

As part of an ongoing study of the operative treatment of colon injuries, 33 patients proved to have colon injury at operation had diagnostic peritoneal lavage as part of their preoperative examination. Gunshot wounds were the cause of injury in 22 (67%) of these patients and stab wounds in 11 patients (33%). The lavage was positive in only 23 (70%) of the 33 patients with operatively proved penetrating injuries of the colon. Lavage was accurate in 16 (73%) of 22 patients with gunshot wounds of the colon and seven (64%) of 11 patients with stab wounds. The overall false-negative rate for this series of patients was 30%. This study indicates that diagnostic peritoneal lavage is relatively inaccurate in the evaluation of colonic injury secondary to penetrating abdominal trauma.

Abdominal Injuries↗
PubMed1983

The importance of the WBC count in peritoneal lavage.

A subset of ten consecutive patients who had sustained penetrating and blunt abdominal trauma came to the hospital with stable vital signs. None had any clinical evidence of intraperitoneal injury. All patients underwent peritoneal lavage, and surgical exploration was performed on the basis of a 100,000/cu mm in the effluent. Substantial intraperitoneal injury was found in nine of ten patients. We suggest that as a further refinement of peritoneal lavage, in addition to RBC count, the WBC count be measured in the effluent fluid and if it is found to be greater than 500/cu mm, surgical exploration be carried out.

Abdominal Injuries↗
PubMed1980

[Preoperative halo-gravity traction in scoliosis (author's transl)].

Clinical experience gained with halo-gravity traction since 1974 is described. The advantage of halo-gravity traction as compared to other types of traction treatment is discussed. Measurements are described, which serve to monitor the treatment and which can be used to predict the final result of the preoperative traction treatment.

Adolescent↗
PubMed1979

[A rare case of so-called idiopathic osteolysis associated with a lymphangioma of the fibula (author's transl)].

In a 70-year old female patient, a so-called idiopathic massive osteolysis with destruction of the proximal right femur within six months, was observed. Histologically, the following conditions were found at the time of resection: a stroma rich in fibres, with the inclusion of blood vessels, as well as an osteoclastic absorption which was still clearly active at the margins, besides marginally defined processes of bone formation. A good functional result without progressing osteolysis at the femur was achieved via a total endoprosthetic replacement of the right hip joint. Within the following three years, polycystic intramedullar osteolysis developed from a slight loosening of the spongiosa in the right head of the fibula. In contrast to the changes which had occurred at the femur, this was a clear case of a rare intraosseous lymphangioma.

Aged↗