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Biomedical subjects

S E Harris

Publications and source records attributed to S E Harris.

At least 109 records · Page 6Linked to original sources
PubMed1983

Serum and growth factor requirements for proliferation of human adrenocortical cells in culture: comparison with bovine adrenocortical cells.

Although bovine adrenocortical cells proliferate readily in cell culture, proliferation of fetal or adult human adrenocortical cells has been observed to be limited and preparation of pure proliferating cultures of human adrenocortical cells has not been reported. The growth requirements of fetal human definitive zone adrenocortical cells in culture were compared to the established requirements of bovine adrenocortical cells. The medium used was 1:1 Ham's F12 and Dulbecco's modified Eagle's medium supplemented with transferrin and insulin. Earlier experiments showed that human cells had a greater proliferative response to horse serum than to fetal bovine serum, whereas the opposite was true for bovine cells. When plated on fibronectin-coated dishes and exposed to varying concentrations of horse serum in the presence of 100 ng/ml fibroblast growth factor (FGF), increasing cell growth was observed up to a serum concentration of 50%. When 50% fetal bovine serum was used instead of horse serum proliferation was less. In contrast, bovine adrenocortical cells showed a maximal proliferative response to either fetal bovine serum or horse serum at 10%. Human adrenocortical cells thus have a very high requirement for serum; 50% is the highest level that may be practically used, but the shape of the dose-response curve suggests that this concentration is still suboptimal. Growth was less in the absence of FGF. Epidermal growth factor can partially substitute for FGF. No response to 100 nM placental lactogen was observed. Less growth was observed when dishes were not coated with fibronectin. The factors present in horse serum that are evidently needed in high amounts by human cells are unknown. Despite this lack of knowledge, use of 50% horse serum enabled long-term growth of human adrenocortical cells that are pure by the criterion of retraction in response to ACTH. Nonadrenocortical cells do not show a retraction response. Such long-term cultures may be useful in studies of long-term regulation of differentiated function, aging, and carcinogenesis.

Adrenal Cortex↗
PubMed1983

Developmental aspects of androgen-dependent mRNA from rat ventral prostate using cloned cDNA.

The androgen-dependence of two mRNAs from rat ventral prostate coding for a 20 000 and an 11 000 dalton translation product has been investigated using complementary DNA cloned in the bacterial plasmid PBR322. One of the cloned insert DNAs from a recombinant plasmid, C-27, arrests the in vitro translation of C2 (Peeters et al. (1980) J. Biol. Chem. 255, 7017-7023). The other cloned insert DNA arrests the translation of a glycoprotein 20 000 daltons in size, with unknown function. The quantities of mRNA coding for the 20 000 and 11 000 dalton translation product were determined by hybridization of 32P-labeled inserts to filter-bound total RNA or poly(A+)-mRNA. Castration caused a decline in both mRNAs of 250-fold over 8 days. Stimulation with androgen of 5-week castrates restored the mRNA levels to 17% of intact for the 20 000 dalton translation product and 31% of intact for the 11 000 dalton translation product. The quantity of the two mRNAs found in the lateral poly(A+)-mRNA was about 1/10 that of the ventral level and the mRNAs were not detectable in the dorsal prostate, seminal vesicle or human prostate poly(A+)-mRNA populations. RNA from the ventral prostates of animals 10-21 days old contained mature levels of complementary sequences, suggesting a form of developmental posttranscriptional regulation for synthesis of the polypeptides which are not synthesized in mature quantities at this stage of development (Heyns et al. (1978) Endocrinology 103, 1090-1095; Kistler et al. (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78, 737-741).

Androgen-Binding Protein↗
PubMed1983

Seminal vesicle secretion IV gene: allelic difference due to a series of 20-base-pair direct tandem repeats within an intron.

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a lambda Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another lambda clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.

Alleles↗
PubMed1983

The seminal vesicle secretion IV gene: detection of S1 nuclease-sensitive sites in supercoiled plasmid pSVS 3.3.

We have investigated the sites sensitive to the S1 nuclease which are presented in a supercoiled plasmid, pSVS 3.3. This plasmid contains the entire transcription unit plus 5'- and 3'-flanking regions of the rat seminal vesicle IV gene on a 3.3 kb Eco RI fragment (Harris et al., 1983). Using a cDNA probe of rat seminal vesicle secretion product IV (SVS IV) mRNA, we have demonstrated S1 nuclease-sensitive sites in the 5'-flanking and in the 3'-flanking regions of SVS IV gene. The site in the 5'flanking region has been mapped to approximately 117 bp upstream from the CAP site. This site at -117 bp can potentially form a cruciform structure as inferred from the presence of inverted repeats in this region (Lilley, 1981; Panayotatos and Wells, 1981). We have now shown such a structure most probably exists. The S1 nuclease-sensitive site in the 3'flanking region is present in an area of highly repeated sequence.

Amino Acid Sequence↗
PubMed1982

Molecular differentiation of the mouse genital tract: protein synthesis in fetal and immature female reproductive tract.

Using two-dimensional gel electrophoresis, approximately 75% of the 192 proteins detected in the fetal mouse genital tract are also detected on fluorograms of immature tissue. There is a progressive increase in the number of qualitatively different proteins synthesized from Day 14 fetal genital tissue to the 17-day-old immature uterus and vagina. Seventy-eight percent of the 180 proteins detectable on fluorograms of proteins having isoelectric points between 4 and 7, and proteins synthesized in the immature female genital tracts, were also detected in fluorograms of fetal tissues. Four of the 12 new proteins appear to be specific to the immature vagina; the remaining 8, specific to the immature uterus.

Animals↗
PubMed1981

Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression.

The abundant class of poly(A+)RNA [poly(A+)RNA11S] from rat seminal vesicle was used to synthesize ds-cDNA11S. The ds-cDNA11S was inserted and cloned into the Pst I site of pBR-322 using E. coli RR1 as host. Colony filter hybridization and restriction mapping was used to demonstrate that a 620 NTP long insert in a plasmid clone (pSV2) represents the almost full length structural gene coding for a precursor to the seminal vesicle secretion protein IV (SVS IV). The entire insert was sequenced and the coding region was matched with the known amino acid sequence. Most of the signal peptide sequence was derived from the DNA sequence. The insert in pSV2 was labelled and used to study the effect of testosterone on the accumulation of mRNA SVS IV. Administration of testosterone to castrated rats resulted in the induction of mRNA SVS IV from a few molecules per cell to levels of over 100,000 after 96 h of hormone treatment.

Animals↗
PubMed1981

Molecular differentiation of the mouse genital tract: serum-free organ culture system for morphological and biochemical correlations.

This report describes a serum-free system for studying the fetal mouse genital tract in organ culture. Using the techniques of isoelectric focusing and gel electrophoresis of polypeptides synthesized by the organ explant in culture, the biochemical integrity of as little as 1 to 2 mg of this tissue was determined during the period of culture. Thus, differentiation of the fetal genital tissue in organ culture can be assessed by both morphological and biochemical criteria.

Animals↗
PubMed1981

Uterine proteins influenced by estrogen exposure. Analysis by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis has been utilized as a technique to investigate uterine protein patterns and protein distribution after estrogen administration. A combined in vivo stimulation, in vitro labeling technique was devised to investigate these questions. Animals were first primed in vivo with estrogen for 2 h to activate the normal early response to hormone and to stimulate protein synthesis. Tissue was removed and protein labeling occurred for 4 h with [35S]methionine. Tissue was then re-exposed in vitro to either estrogen or saline for an additional 1 h. Subfractionation of the uterine tissue and analysis of labeled proteins by two-dimensional gel electrophoresis has indicated the disappearance of some proteins from the cytoplasmic compartment and the appearance of other proteins in the nuclear fraction of estrogen-exposed samples. These findings suggest that in the mouse uterus, estrogen influences protein synthesis and also a redistribution of these proteins into various cellular fractions where they may act to elicit estrogen action.

Animals↗
PubMed1981

Effect of testosterone propionate on protein synthesis by two-dimensional electrophoresis in rat ventral prostate.

Two-dimensional polyacrylamide gel electrophoresis has been used to map the pattern of protein synthesis in rat ventral prostate from both castrate and testosterone treated animals. Proteins were detected by fluorography of 35S-methionine incorporation. In the soluble or cytosol fraction, four major proteins that have molecular weights less than 25,000 and isoelectric points (pI) less then 6.0 and one than has a molecular weight above 30,000 and pI 6.6 are under androgen control. One of these proteins with molecular weight 12,000 and pI 5.4 also appears in the microsomal fraction. Using nonequilibrium pH gradient electrophoresis, soluble proteins of pI greater than 7.0 were investigated. One major protein of 20-25,000 daltons and pI 8.0 appears to be androgen dependent. The ventral prostate from castrate synthesized proteins which are not detectable in androgen stimulated castrates.

Animals↗
PubMed1980

Purification of major abundance class of poly(A+)-RNA from rat ventral prostate.

Poly(A+)--RNA from rat ventral prostate was isolated using oligo(dT)-cellulose chromatography. 45% of the total poly(A+)--RNA was a single peak at 10S as demonstrated by centrifugation in a 5-20% sucrose gradient containing 1% SDS. By using complementary DNA probes, it was shown that the 10S RNA contained the major abundance class of poly(A+)--RNA. Denaturing agarose-gel analysis revealed 2 major bands in the 10S poly(A+)--RNA preparation approx. 600 NT and 500 NT (NT = nucleotides) long, resp. Double-stranded 32 P-DNAs complementary to light side and heavy side of the 10S poly(A+)--RNA peak were synthesized and isolated using reverse transcriptase and hydroxyapatite (HAP) chromatography. Approx. 40% of the first strand of the cDNAs were converted to double-stranded structures with a Tm of 88 degrees C. HAP purified double-stranded material was 92% resistant to S1 nuclease. the DNA--DNA reannealing profile of double stranded 32 P-cDNA enriched for the 500 NT band gave a Cot 1/2 of approximately 7 X 10(-4) moles X sec X 1(-1) indicating a complexity for this enriched synthetic gene of 500-600 nucleotide pairs (NTP).

Animals↗
PubMed1980

Effect of spironolactone on androgen-dependent proteins in the ventral prostate of the rat.

The effect of spironolactone on five androgen-dependent proteins in the ventral prostate of the rat was investigated by two-dimensional gel electrophoresis. Spironolactone was given to intact male, castrated and androgen-stimulated castrated rats. It has been shown that spironolactone had no influence on the synthesis or accumulation of the androgen-dependent proteins in intact animals. However, spironolactone suppressed the restoration of the major androgen-dependent protein of low molecular weight in castrated rats given testosterone. The mechanism by which spironolactone exerts its anti-androgenic activity was shown to be unrelated to its capacity to inhibit the synthesis or accumulation of the five androgen-dependent proteins studied in this investigation.

Animals↗
PubMed1980

In vitro translation of major abundant class poly(A+)-mRNA from rat seminal vesicle.

Total poly (A)-containing messenger RNA [poly(A)-mRNAT] was isolated from seminal vesicles of adult male rats. By preparative 5%-20% linear sucrose gradient centrifugation in 1% SDS, a single peak was seen in the 11S region [poly(A)-mRNA11S]. Poly (A)-mRNAT and poly(A)-mRNA11S were translated in the wheat germ S-30 in vitro translation system. Laemmli-SDS-polyacrylamide slab gel electrophoresis of translation products of poly(A)-mRNAT revealed three major protein bands at approximately 18,500, 15,000, and 14,000 daltons. In vitro translation of poly(A)-mRNA11S across the 11S peak revealed partial purification of the mRNAs for the three predominant proteins. The lower molecular weight part of the 11S peak enriched for the mRNAs coding for the 15,000 and 14,000 dalton polypeptides, and the higher molecular weight side of the peak enriched for the 18,500 dalton mRNA. The highly abundant class of mRNA from rat seminal vesicle can be purified with one sucrose gradient centrifugation.

Animals↗
PubMed1979

Isolation and partial purification of the major abundant class rat seminal vesicle poly(A+)-messenger RNA.

Total poly(A(+))-RNA (poly(A(+))-RNA(tot)) was isolated from rat seminal vesicle and its size distribution determined by 70% formamide 5-25% sucrose density analysis. One major peak was resolved in the 10-13 S region and accounted for approximately 35% of the total poly(A(+))-RNA applied. Preparative 1% SDS, 5-20% linear sucrose density gradients also resolved a single major peak in the 11S region (poly(A(+))(11S). Analysis of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) under denaturing conditions on 2% agarose gel electrophoresis demonstrated two major components in both poly(A(+))-RNA populations. Size estimations for these components are 620 and 540 NT respectively. (3)H-cDNA was made to both poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S). Back-hybridization of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) to their respective (3)H-cDNA revealed a highly abundant class representing 41% and 85% of the sequences in their respective (3)H-cDNA's. The highly abundant class corresponded to 3-5 sequences present in 30,000-50,000 copies/cell. Invitro translation of poly(A(+))-RNA(11S) resulted in two major polypeptides coded for by the 620 NT long and 540 NT long poly(A(+))-RNA respectively.Images

Animals↗
PubMed1978

Schizophrenics' mutual glance patterns.

The schizophrenic patient is often quite incommunicative about his current or past interpersonal relationships. Many workers feel that interviews with the schizophrenic's parents are also unreliable means of getting direct verbal information. Such pessimism is based on their experience that parents of schizophrenics often distort or delete information because of a concern with appearances, an inability to communicate adequately, or an investment in maintaining the patient status of their offspring. Difficulty in getting verbal data limits the therapist's understanding of the schizophrenic and his relationship with people. Observation of implicit communications through gestural interaction provides an important alternate means of getting information about interpersonal relationships which does not necessarily require the cooperation of the patient or his parents. As research in gestural communication improves, it should be possible to attempt a more detailed investigation of the interaction between the schizophrenic patients and others. Through the use of gestural or other implicitly communicative indices, such qualities as seductiveness, hostility, dependency, dominance, and others may ultimately prove to be measurable. Research has demonstrated that gestural behavior as a means of imlicit communication is indicative, in a gross sense, of the quality of interaction between people (Ekman, 1965; Exline and Winter, 1965). This study is concerned with mutual glances--moments in which two people look directly at each other at the same time so that their eyes meet.

Adult↗