Biomedical subjects

J Tenhunen

Publications and source records attributed to J Tenhunen.

26 records · Page 2Linked to original sources

PubMed1991

Increased steroid hormone secretion in mouse Leydig tumor cells after induction of cholesterol translocation by sphingomyelin degradation.

The effects of sphingomyelin degradation on [3H]cholesterol transfer from the cell surface to mitochondria were examined in mouse Leydig tumor cells. These cells were used since they utilize cholesterol for steroid hormone synthesis in the mitochondria, and also possess acyl-CoA: cholesterol acyl transferase (ACAT) activity in the endoplasmic reticulum. Exposure of glutaraldehyde-fixed mouse Leydig tumor cells to sphingomyelinase (50 mU/ml, 60 min) resulted in the degradation of about 50% of cell sphingomyelin, suggesting that only half of the sphingomyelin mass in these cells was located in the exoleaflet of the plasma membrane. The partial sphingomyelin degradation resulted in the translocation of cellular unesterified [3H]cholesterol from plasma membranes (cholesterol oxidase-susceptible) to intracellular compartments (oxidase-resistant). The fraction of [3H]cholesterol that was translocated, i.e., between 20 and 50%, varied with different [3H]cholesterol-labeling methods. Cholesterol translocation induced by sphingomyelin degradation subsequently led to the stimulation of ACAT activity, suggesting that a fraction of cell surface cholesterol was transported to the endoplasmic reticulum. The sphingomyelinase-induced [3H]cholesterol flow from the cell surface to the cell interior was also in part directed to the mitochondria, as evidenced by the increased secretion of [3H]steroid hormones. In addition, the cyclic AMP-induced activation of steroidogenesis was further enhanced by the sphingomyelinase-induced cholesterol translocation. Based on the current results, it seems evident that a significant portion of the translocated [3H]cholesterol made its way from plasma membranes into the mitochondria for steroidogenesis.

Animals↗

PubMed1990

Effects of sphingomyelin degradation on cholesterol mobilization and efflux to high-density lipoproteins in cultured fibroblasts.

The hydrolysis of sphingomyelin from cellular plasma membranes imposes many consequences on cellular cholesterol homeostasis by causing a rapid and dramatic redistribution of plasma membrane cholesterol within the cells (Slotte, J.P. and Bierman, E.L. (1988) Biochem. J. 250, 653-658). The objective of this study was to examine the effects of an extracellular cholesterol acceptor on the directions of the sphingomyelinase-induced cholesterol flow in cultured fibroblasts. We have used HDL3 as a physiological acceptor for cholesterol, and measured the effects of sphingomyelin hydrolysis on efflux and endogenous esterification of cellular [3H]cholesterol. Treatment of cells with sphingomyelinase did induce a dramatically increased esterification of plasma-membrane-derived [3H]cholesterol. The presence of HDL3 in the medium (100 micrograms/ml) did not prevent or reduce the extent of the sphingomyelinase-induced cellular esterification of [3H]cholesterol. Degradation of cellular sphingomyelin (75% hydrolysis) also did not enhance the rate of [3H]cholesterol efflux from the plasma membranes to HDL3. In addition, we also observed that the degradation of sphingomyelin in the HDL3 particles (complete degradation) did not change the apparent rate of [3H]cholesterol transfer from HDL3 to the cells. These findings together indicate that hydrolysis of sphingomyelin did not markedly affect the rates of cholesterol surface transfer between HDL3 and cells. By whatever mechanism cholesterol is forced to be translocated from the plasma membranes subsequent to the degradation of sphingomyelin, it appears that the sterol flow is specifically directed towards the interior of the cells.

Cholesterol↗

PubMed1990

A solution hybridization method for quantification of mRNAs: determining the amount and stability of oncogene mRNA.

A solution hybridization method for the quantification of specific mRNAs is described. This assay utilizes complementary RNA probes prepared by in vitro transcription, sandwich hybridization in solution, and affinity-based hybrid collection. The possibility of using this method for crude biological samples without purifying mRNAs makes it ideal when accurate quantification of multiple samples is needed. Human N-myc oncogene transcript was used as a model and as little as 0.24 pg (2 X 10(5) molecules) of N-myc mRNA could be detected. Using this assay it was shown that human neuroblastoma IMR-32 cells contain approximately 500 N-myc mRNA molecules per cell having a half-life of approximately 35 min.

DNA Probes↗

PubMed1989

Hydrolysis of single-stranded RNA in aqueous solutions--effect on quantitative hybridizations.

Single stranded RNA is non-enzymatically hydrolysed in aqueous solutions at neutral pH at elevated temperatures. This hydrolysis causes practical problems in different hybridization procedures. Synthetic cRNA transcribed from the human p53 oncogene was found to be partially destroyed after 6 hours and completely destroyed after overnight incubation at 60 degrees C. At lower temperatures the cRNA was preserved intact in spite of overnight incubation, but at higher temperatures (80 degrees C) the degradation occurred in less than 2 hours. The effect of the hydrolysis on hybridization results was studied by measuring in solution the hybridization kinetics of the cRNAs of another human oncogene, N-myc. It is obvious that conditions generally used for DNA hybridizations cannot be utilized for RNA in quantitative studies.

False Negative Reactions↗

PubMed1988

Quantification of polymerase chain reaction products by affinity-based hybrid collection.

We have used oligonucleotides modified with biotin in the 5'-end as primers in the polymerase chain reaction (PCR)-amplification. This results in the synthesis of 5'-biotinylated DNA molecules, which are detected by hybridization to a labelled probe in solution. The formed hybrids are collected on an avidin-matrix by mediation of the biotin residue of the target molecules. The affinity-based hybrid collection method is quantitative and makes it possible to measure the amount of DNA produced in the PCR-amplification. At low concentrations of template the efficiency of the process is close to 100%, making it possible to detect the presence of a few molecules of target DNA in 25 cycles. With high template concentrations the efficiency of the process is low.

Affinity Labels↗

PubMed1988

Screening of premalignant cervical lesions for HPV 16 DNA by sandwich and in situ hybridization techniques.

A series of 97 cervical smears and 69 directed punch biopsies derived from 84 consecutive women prospectively followed-up for cervical HPV (human papillomavirus) infections were studied using the sandwich hybridization and in situ hybridization techniques with HPV 16 DNA probes. The aim was to test the sensitivity and applicability of these two techniques in routine diagnosis of cervical HPV infections from smears. As a measure of specimen adequacy, the number of cells recovered in the cervical scrape was determined along with HPV 16 DNA in the sandwich hybridization test using human pro-alpha 2(I)-collagen gene probe. CIN (cervical intraepithelial neoplasia) was suggested in 56% of the patients by the Pap smear, and disclosed in 65% of the biopsies. HPV 16 DNA was present in 57% of cervical scrapes consistent with CIN, i.e., were of Pap smear classes III or IV. Forty percent of the scrapes not suggestive of CIN, i.e., Pap smear classes I or II, also contained HPV 16 DNA. The detection rate for HPV 16 DNA of the sandwich hybridization method was 89% of that of the in situ method in adequate scrapes, but only 43% in cell-poor specimens. The number of HPV 16 DNA-positive scrapes as compared with the total number of diagnoses obtained by studying also the biopsies was 31/36 (69 patients). The results indicate that the cervical scrape as a noninvasive specimen is applicable for screening of cervical HPV infections, and it can be studied with acceptable sensitivity by the rapid sandwich hybridization technique. However, if a punch biopsy is indicated it should be studied using the in situ hybridization technique that allows more sensitive detection of HPV DNA than any other hybridization method and enables the analysis of several HPV types in the same sample instead of only one HPV type in the scrapes.

DNA, Viral↗

PubMed1986

Fimbriation and P-antigen recognition of Escherichia coli strains harbouring mutated recombinant plasmids encoding fimbrial adhesins of the uropathogenic E. coli strain KS71.

Deletion mutants of recombinant plasmids encoding the KS71B fimbrial antigens of the uropathogenic Escherichia coli strain KS71 (O4:K12) were constructed. The effects of these mutations were tested by transforming the mutated plasmids into non-fimbriated E. coli HB101 cells and testing the transformants for fimbriation and haemagglutination. A deletion transcriptionally upstream from the fimbrial subunit gene increased the expression of KS71B fimbriae. Deletion of the fimbrial subunit gene resulted in non-fimbriated but haemagglutinating transformants, whereas a deletion 6 kb transcriptionally downstream from the subunit gene resulted in non-haemagglutinating but fimbriate transformants, indicating that fimbriation and haemagglutination were genetically separable. We also present evidence suggesting that the fimbrillin and haemagglutinin are physically associated in the wild-type KS71 strain.

Adhesins, Escherichia coli↗

PubMed1986

Analysis of P fimbriae on Escherichia coli O2, O4, and O6 strains by immunoprecipitation.

P fimbriae on Escherichia coli O2, O4, and O6 strains were analyzed by immunoprecipitation. Fimbrial extracts were prepared from a total of 35 strains and tested for precipitation with four anti-P-fimbria sera. The overall fimbrial composition of the strains was related to the O:K:H serotype, and two to three P fimbrial variants per strain were found on most of the O4 and some of the O6 strains. The O2 strains, in contrast, showed only one antigenic variant of P fimbriae per strain, which was serologically unrelated to those of the O4 and O6 strains. The results stress the multiplicity and serological complexity of E. coli P fimbriae.

Animals↗