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Biomedical subjects

S Ackerman

Publications and source records attributed to S Ackerman.

At least 37 records · Page 2Linked to original sources
PubMed1990

Transcription factor IIA of wheat and human interacts similarly with the adenovirus-2 major late promoter.

Transcription factor IIA (TFIIA) is a necessary component of many RNA polymerase II transcription complexes. Assembly of the transcription complex begins when TFIIA interacts with the promoter. We have previously purified wheat germ TFIIA to homogeneity and demonstrated that it substitutes for human TFIIA in a human in vitro transcription system which utilizes the adenovirus-2 major late promoter (Ad-2 MLP). We now show, by gel retardation assays, that wheat TFIIA interacts with the Ad-2 MLP. Extensively purified human (HeLa) TFIIA interacts with the Ad-2 MLP similarly. Both wheat and human TFIIA interact with a DNA fragment comprising the minimal promoter region (-51/+32) but not with upstream or downstream regions. With both TFIIAs multiple complexes form; the fastest wheat TFIIA/DNA complex appears to be larger than the corresponding human TFIIA/DNA complex. Limited point mutation analysis of the Ad-2 MLP demonstrates that changes at -30 (TATAA region), +1, and -1 diminish TFIIA binding, but a change at -40 does not. DNA footprint analysis of this region is not definitive, but does indicate that following TFIIA binding there are changes in the pattern of hypersensitive sites.

Adenoviridae↗
PubMed1990

Early events of RNA polymerase II transcription initiation.

We have investigated the earliest stages of assembly of an RNA polymerase II transcription complex. General transcription factors from HeLa cells were partially purified and assayed using the adenovirus-2 major late promoter. Preincubation of either all the transcription factors (TF) with the DNA or only the subset consisting of TFIIA, TFIID, and DNA overcame the 15-20 min lag normally observed. The kinetics demonstrate that TFIIA first interacts with the template over a 5 min. period, and then TFIID interacts with the IIA:DNA complex over a 2 min. period. The remainder of the necessary transcription factors then interact with the IIA:IID:DNA complex. There are apparently interactions between IIA and IID, as a pre-incubation of these factors (without DNA) overcomes the lag period. Both IIA:DNA and IIA:DNA:IID interactions are temperature sensitive, resulting in slower kinetics at 0 degree C. Thus, the kinetics of transcription involve activation processes in addition to DNA binding.

Adenoviridae↗
PubMed1989

RNA polymerase II transcription complexes are destabilized by ATP or GTP.

In vitro transcription by RNA polymerase II requires hydrolysis of the beta-gamma bond of ATP after the transcription complex forms, prior to RNA synthesis. It was observed that the presence of ATP during transcription complex formation inhibits subsequent transcription when the remaining 3 rNTPs are added. We now report that ATP or GTP inhibits transcription if either is present during transcription complex formation to added to preformed complexes. This inhibition is not due to purine rNTP degradation and occurs if as little as 2 mM ATP or 50 mM GTP is added to forming or preformed complexes. Deoxy derivatives of ATP inhibit similarly. AMP-PNP, a beta-gamma imido derivative, neither satisfies the energy requirement nor inhibits transcription if added to incubations of forming or of preformed transcription complexes.

Adenosine Triphosphate↗
PubMed1989

Drug inhibitors of RNA polymerase II transcription.

Transcription by RNA polymerase II occurs after formation of a transcription complex. This complex is assembled in stages by the interaction of transcription factors with the template and/or with each other. We report on the ability of six drugs to inhibit the assembly of the RNA polymerase II transcription complex. Assembly of the complex on the adenovirus major late promoter requires several transcription factors. The normal assembly process requires that the DNA first interact with TFIIA, then with TFIID, and finally with at least four additional transcription factors (one of which is RNA polymerase II). We observed that streptolydigin (10 micrograms/ml) inhibits association of ILA and IID, and at higher concentrations (100 micrograms/ml) inhibits that IIA/IID complex from binding to DNA. Streptovaricin (100 micrograms/ml) appears to inhibit the IIA/IID interaction with DNA and prevents reinitiation (at 500 micrograms/ml). Adriamycin (1 microgram/ml) inhibits the interaction of TFIID with the IIA/DNA complex and inhibits an additional event immediately prior to, or during, elongation. Daunorubicin may be an elongation inhibitor. Heparin at 10 micrograms/ml inhibits further assembly after the IIA/IID/DNA complex has formed, and at 100 micrograms/ml also inhibits a late event in the assembly process and blocks reinitiation. Rifamycin AF/013 (100 micrograms/ml) inhibits the early events necessary to form the IIA/IID/DNA complex and (at 10 micrograms/ml) an assembly event following formation of the IIA/IID/DNA complex. Therefore, these compounds should be useful as probes for further examination of the assembly process.

Aminoglycosides↗
PubMed1988

Fertilizing potential of acrosome-defective sperm following microsurgical injection into eggs.

Ejaculates from three infertile men were examined ultrastructurally and found to include a high number of amorphous acrosomeless spermatozoa. Two of the patient's spermatozoa exhibited the typical characteristics of round-head syndrome--spherical-shaped heads completely absent of acrosome and postacrosomal sheath. The semen of the third patient was found to contain a mixture of round-headed and irregularly shaped acrosomeless sperm and a small percentage of normal acrosome-intact sperm. Previous studies have shown that acrosomeless sperm do not have the ability to bind or penetrate zona-free hamster eggs (Weissenberg et al., Syms et al.). In an attempt to determine if such amorphous sperm are capable of decondensation and pronuclear formation, sperm of all three men were microsurgically injected into zona-intact hamster eggs. All of the sperm injected were found to be capable of decondensation or pronuclear formation, suggesting that if the inability to penetrate an egg is bypassed, the sperm of these infertile men are capable of participating in the early events of fertilization.

Acrosome↗
PubMed1988

Effects of antibiotics on RNA polymerase III transcription.

We investigated the effects of six drugs on an RNA polymerase III in vitro transcription system. Adriamycin, daunorubicin, heparin, rifamycin AF/013, streptolydigin, and streptovaricin all inhibit RNA synthesis from a tRNA gene or the adenovirus 2 (AD2) VA1 RNA gene. The completed RNA polymerase III transcription complex is formed by the sequential, ordered addition of protein factors. Although both genes reportedly use the same transcription fractions for in vitro RNA synthesis, some of these drugs interfere differentially with these genes. A drug concentration that inhibits transcription from one gene may not inhibit transcription from the other gene. Adriamycin seems to block transcription if added between the binding of the individual transcription fractions. Daunorubicin appears to inhibit VA transcription only if added prior to both transcription fractions, but inhibits tRNA synthesis before and during transcription factor binding. Heparin blocks both genes between factors binding to DNA and after factor binding. Rifamycin blocks VA synthesis more effectively than tRNA synthesis. Streptolydigin blocks transcription of both genes. Streptovaricin probably blocks transcription by inhibiting early transcription complex assembly events. These drugs appear useful as appropriate probes to investigate transcription complexes since several discriminate between complexes formed on different genes during the assembly process.

Adenoviruses, Human↗
PubMed1987

Kinetic immunonephelometric determination of protein concentrations in follicular fluid.

Nonsteroidal biochemical markers of oocyte maturity could presumably aid in oocyte selection and in the timing of insemination for in vitro fertilization. We assessed the usefulness of six potential markers found in follicular fluid. We used kinetic immunonephelometry to measure concentrations of alpha 1-antitrypsin, alpha 2-macroglobulin, antithrombin III, ceruloplasmin, fibrinogen, and plasminogen in 53 fluids and the corresponding plasma from 20 women undergoing in vitro fertilization. Specimens were obtained from both mature and immature follicles from each woman. The respective protein concentrations in follicular fluid could be assayed with a between-assay CV of 1.5% to 3.2%. Analytical-recovery studies indicated that only fibrinogen and antithrombin III concentrations were altered (by 120% and 75%, respectively) during aspiration of the follicle into saline. All protein concentrations were significantly increased in mature follicles, as were the follicular fluid:plasma concentration ratios for alpha 1-antitrypsin, antithrombin III, and ceruloplasmin.

Antithrombin III↗
PubMed1987

Therapeutic insemination.

Except in special circumstances, therapeutic insemination with a husband's sample has a low success rate. Couples in whom oligozoospermia has been identified as the principal cause of infertility do not benefit from therapeutic insemination by husband. Because of this low success rate, intrauterine insemination to provide sperm in closer proximity to the egg has become popular, but intrauterine insemination also has a low success rate. We suggest that intrauterine insemination should be approached aggressively in cases of male factor infertility. The recipient should be stimulated to enhance egg production and closely monitored for ovulation. A semen specimen of not less than 1 X 10(6) motile sperm with antibiotics added should be placed in the uterus the day after ovulation. If no pregnancies occur within four cycles, alternate approaches should be considered. Therapeutic insemination by donor involves careful donor selection to avoid inheritance of malformations and familial diseases. Because of the possibilities of sexually transmitted diseases, careful and repeated screening should be conducted. A complete sexual history should be obtained, and donors should be excluded if they have had any homosexual contact since 1978, if they have been an intravenous drug user, if they come from a geographic area where the sex ratio of AIDS is close to 1:1, or if they have recently had multiple sexual partners. A permanent record preserving the confidentiality but allowing the tracing of genetic anomalies, even if not present at birth, should be kept.

Cell Separation↗
PubMed1986

The epidemiology of inflammatory bowel disease in Cape Town 1980-1984.

Patients with inflammatory bowel disease diagnosed in the greater Cape Town area between the years 1980 and 1984 inclusive were studied to establish the incidence and presenting features of these diseases. Case details were obtained from the records of the Groote Schuur Hospital group and Tygerberg Hospital as well as from questionnaires completed by 97% of 731 medical practitioners in the area. There were 134 patients with Crohn's disease, 197 with ulcerative colitis and 16 with indeterminate colitis. The incidences of Crohn's disease in the coloured, white and black population groups were calculated to be 1.8, 2.6 and 0.3/100 000 per year respectively and those for ulcerative colitis 1.9, 5.0 and 0.6/100 000 respectively. Among Jews the incidence was higher for both Crohn's disease (10.4/100 000) and ulcerative colitis (17.0/100 000). Of patients with ulcerative colitis 35% had limited proctitis, 26% proctosigmoiditis, 12% left-sided disease and 27% total colitis. Of patients with Crohn's disease 27% had colonic involvement alone, 37% ileocolitis and 36% small-bowel involvement alone. The incidence of inflammatory bowel disease has increased in the greater Cape Town area over the last 10 years, while the pattern of disease has remained similar to that previously reported for this geographical area.

Adolescent↗
PubMed1985

Male factor evaluation in in vitro fertilization: Norfolk experience.

Thirty-three patients from the in vitro fertilization (IVF) program at Norfolk are critically reviewed. A battery of tests was designed and an endocrine investigation was carried out on these patients. The fertilization rate for preovulatory oocytes was lower than in the normal male population (39.6% versus 88.6%). When total concentration of sperm with rapidly progressive motility was less than 6 X 10(5), to fertilize several eggs together the fertilization rate was zero. No fertilization was obtained when the number of sperm with rapidly progressive motility recovered after the separation was less than 1.5 X 10(6). The hamster zona-free oocyte penetration test correlated well with the human IVF system. The other parameters investigated did not show good correlation. When fertilization was achieved, the results of the IVF procedure in the series reviewed rendered a 30.8% pregnancy rate per transfer in 26 transfers. Fifty percent of the pregnancies were normal (either ongoing or delivered). Thirty-seven percent were preclinical miscarriages, and 12.5% were clinical abortions. In the abnormal male population, higher concentrations of sperm per egg should be used for insemination for achievement of optimum fertilization rates. Once fertilization is obtained, the results do not differ substantially from the IVF population at large.

Animals↗
PubMed1983

RNA polymerase II ternary transcription complexes generated in vitro.

Ternary transcription complexes have been formed with a HeLa cell extract, a specific DNA template, and nucleoside triphosphates. The assay depends on the formation of sarkosyl-resistant initiation complexes which contain RNA polymerase II, template DNA, and radioactive nucleoside triphosphates. Separation from the other elements in the in vitro reaction is achieved by electrophoresis in agarose - 0.25% sarkosyl gels. The mobility of the ternary complexes in this system cannot be distinguished from naked DNA. Formation of this complex is dependent on all parameters necessary for faithful in vitro transcription. Complexes are formed with both the plasmid vector and the specific adenovirus DNA insert containing a eucaryotic promoter. The formation of the complex on the eucaryotic DNA is sequence-dependent. An undecaribonucleotide predicted from the template DNA sequence remains associated with the DNA in the ternary complex and can be isolated if the chain terminator 3'-0-methyl GTP is used, or after T1 ribonuclease treatment of the RNA, or if exogenous GTP is omitted from the in vitro reaction. This oligonucleotide is not detected in association with the plasmid vector. Phosphocellulose fractionation of the extract indicates that at least one of the column fractions required for faithful runoff transcription is required for complex formation. A large molar excess of abortive initiation events was detected relative to the level of productive transcription events, indicating a 40-fold higher efficiency of transcription initiation vs. elongation.

DNA-Directed RNA Polymerases↗
PubMed1983

Mechanism of action of DRB. III. Effect on specific in vitro initiation of transcription.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole, an adenosine analogue, has been used previously as an inhibitor of heterogeneous nuclear and messenger RNA synthesis. In an in vitro transcriptional system, we have detected inhibition of synthesis of full-length runoff RNAs at concentrations at which in vivo mRNA synthesis is inhibited. By hybridization of RNA synthesized in vitro to single-stranded DNA and gel analysis, we were able to reduce the background of the transcription reaction, detect DRB-induced inhibition of full-length runoff RNAs and DRB-insensitive transcription of short RNAs. To establish further the effect of DRB on initiation of transcription, preincubation experiments with template, whole cell extract and two initial nucleotides of the transcript were performed. Elongation was then measured as discrete-sized RNAs transcribed from the truncated template after addition of the other triphosphates (one of them labeled), in the presence or absence of DRB. An effect on initiation but not on elongation or termination was detected. Fingerprint analysis of these runoff RNAs indicates that the labeling of U in the presence of DRB is uniform throughout the molecule. A model to explain a novel interpretation of the action of DRB is presented.

Adenoviridae↗
PubMed1983

The management of obesity.

The ineffectiveness of most weight-reduction protocols is notorious. Improvement of current management depends on individualizing the approach and recognizing the importance of the chronology of onset and the duration of the obesity.

Adipose Tissue↗