Biomedical subjects

T C Wood

Publications and source records attributed to T C Wood.

At least 37 records · Page 2Linked to original sources

PubMed1997

Phenol sulfotransferase pharmacogenetics in humans: association of common SULT1A1 alleles with TS PST phenotype.

The phenol sulfotransferases (PSTs) catalyze the sulfation of both small planar phenols and phenolic monoamines. Three highly homologous PST genes, SULT1A1, SULT1A2, and SULT1A3, are known to exist in humans. The prototypic biochemical phenotype associated with the enzyme encoded by SULT1A1 is the thermal stable (TS) sulfation of 4 microM 4-nitrophenol (TS PST activity). Biochemical pharmacogenetic studies have demonstrated that individual variation in both TS PST activity and thermal stability in humans are inherited. As a step toward understanding molecular mechanisms responsible for the genetic regulation of PSTs in humans, we report here common SULT1A1 nucleotide polymorphisms that are associated with phenotypic variation in both platelet TS PST activity and thermal stability. When 905 human subjects were phenotyped for platelet TS PST activity and thermal stability, activity varied more than 50-fold, and thermal stability varied over 10-fold. DNA was isolated from the blood of 33 of these subjects selected on the basis of "extreme" TS PST phenotypes: high activity and high thermal stability; low activity and low thermal stability; or low activity and high thermal stability. These 33 subjects were genotyped for SULT1A1 by PCR amplification and sequencing of the entire open reading frame (ORF) as well as approximately 1 kb of intron DNA sequence. One common allele, SULT1A1*2, was uniformly associated with both very low TS PST activity and low thermal stability. The allele frequency of SULT1A1*2 in a randomly selected population sample of 150 Caucasian blood donors was 0.31 (31%), indicating that approximately 9% of this population would be homozygous for that allele.

Alleles↗

PubMed1997

Follicle-oocyte atresia and temporal taphonomy in cold-stored domestic cat ovaries.

In vitro oocyte maturation followed by in vitro fertilization (IVM/IVF) success in the domestic cat remains inferior to commonly studied livestock or laboratory species. The objectives here were (1) to histologically assess atresia status of freshly excised follicle/oocyte complexes, and (2) to evaluate taphonomic change (deterioration after excision) of these complexes after ovarian cold storage for up to 48 h. After excision of 50 ovarian pairs, one ovary was preserved immediately and the other stored in phosphate buffered saline (4 degrees C) for 4, 8, 12, 24, or 48 h before fixation and examination. Ovaries were classified as luteal if prominent corpora lutea (CL) were present or as follicular if antral follicles and no CL were present. Two classes of follicle-oocyte complexes (preantral and antral) were microscopically evaluated. Of the 2,280 complexes examined, 64.3% demonstrated clear evidence of slight to severe degeneration, with various stages being described and photographed for the first time. There was no histological evidence indicating distinctive morphological differences between oocytes recovered from follicular versus luteal donors. Storage of whole ovaries in cold saline inhibited taphonomic changes for 48 h after excision. In summary, there is marked variability in the number and quality of follicle populations in cat ovaries. A high percentage of full-sized follicular oocytes are undergoing atresia at any given time. However, additional gross degeneration as a result of cold-storage appears modest for up to 48 h. Nonetheless, this high level of natural atresia in the cat likely contributes to comparatively lower IVM/IVF success than in other species.

Animals↗

PubMed1997

DNA degradation in mural granulosa cells of non- and slightly atretic follicles of fresh and cold-stored domestic cat ovaries.

Apoptosis of granulosa cells is associated with follicular atresia and may occur before atresia becomes morphologically evident. Detection of DNA fragmentation by in situ end-labeling (ISEL) with terminal transferase allows the histological assessment of apoptotic cells on conventional histological sections. Degradation of DNA also may occur after prolonged cold storage of ovaries caused by the release of lysosomal enzymes. The objectives of this study were to assess follicle atresia and the impact of cold storage for 8, 12, 24, and 48 hr after ovarian excision by assessing DNA degradation in mural granulosa cells of cat ovaries. Follicles were distinguished by morphological criteria as nonatretic (NA), slightly atretic (SA), or atretic, and the mean number (+/-SEM) of granulosa cells labeled by ISEL was determined. About 50% of follicles showed some sign of atresia independent from the stage of the reproductive cycle of the ovarian donor. Number of ISEL-stained granulosa cells for NA and SA, freshly collected follicles was 7.5 +/- 0.6 and 9.3 +/- 0.8 cells/field, respectively, compared to 16.2 +/- 0.8 cells/field in the wall of atretic follicles (P < 0.001). Fresh NA follicles from luteal phase ovaries had more (P < 0.05) labeled granulosa cells (9.2 +/- 0.7 cells/field) than measured in follicles of cats in a follicular phase (5.7 +/- 0.7). During cold storage, DNA degradation began within 12 hr (NA, 12.2 +/- 0.7 cells/field; SA, 13.3 +/- 0.5), both values being different (P < 0.05) from fresh controls. By 24 hr, DNA degradation was at the level of a positive control subjected to DNAse treatment. In summary, results reveal that granulosa cell DNA degeneration precedes the loss of developmental capacity of cat oocytes during atresia and postexcision storage. Finding irreversible changes in granulosa cell DNA after storage of cat ovaries for > 12 hr may be important for developing oocyte rescue protocols for rare felids in cases where prolonged storage and transport may be required.

Animals↗

PubMed1997

Effect of the quality of the cumulus-oocyte complex in the domestic cat on the ability of oocytes to mature, fertilize and develop into blastocysts in vitro.

Immature cumulus-oocyte complexes (COCs) were recovered from freshly excised domestic cat ovaries and graded at a magnification of x40 for the condition of the cumulus oophorus of the oocyte cytoplasm. Grade I and II COCs were those with a uniformly dark cytoplasm and a readily identifiable, eccentrically located germinal vesicle. Grade I COCs had five or more cumulus oophorus cell layers, whereas grade II complements had less than five cell layers. Grade III and IV COCs were those undergoing progressive stages of oocyte cytoplasmic deterioration indicated by transparency or mosaic fragmentation and partial-to-complete loss of cumulus oophorus cells. In Expt 1, 699 oocytes were cultured for maturation and fertilization in vitro. More (P < 0.05) oocytes from grade I COCs matured (59.3%) and fertilized (29.7%) than from all other grades. Maturation and fertilization success did not differ (P > 0.05) for grade II (32.4, 11.6%, respectively) and grade III (21.9, 5.1%) oocytes, but these values were superior (P < 0.05) to those of grade IV (5.1, 1.4%). In Expt 2, 1040 COCs were graded, cultured for maturation and then inseminated. Of grade I oocytes, 24.4% developed into blastocysts compared with only 5.3% of grade II oocytes (P < 0.05). In general, oocytes from grade III and IV COCs were incapable of cleaving or growing in vitro. Of the 1739 COCs collected for both experiments, 12.3% met grade I criteria, the only category that provided consistent maturation, fertilization and development to blastocyst stage in vitro. In summary, a highly heterogeneous population of cumulus-oocyte complexes can be separated in the cat on the basis of grossly apparent morphological characteristics that, in turn, reflect functional differences in the ability of oocytes to mature, fertilize and develop in vitro.

Animals↗

PubMed1997

Sulfation and sulfotransferases 1: Sulfotransferase molecular biology: cDNAs and genes.

Sulfotransferase (ST) enzymes catalyze the sulfate conjugation of many hormones, neurotransmitters, drugs, and xenobiotic compounds. These reactions result in enhanced renal excretion of the sulfate-conjugated reaction products, but they can also lead to the formation of "bioactivated" metabolites. ST enzymes are members of an emerging gene superfamily that presently includes phenol ST (PST), hydroxysteroid ST (HSST), and, in plants, flavonol ST (FST) "families," members of which share at least 45% amino acid sequence identity. These families can be further subdivided into "subfamilies" that are at least 60% identical in amino acid sequence. For example, the PST family includes both PST and estrogen ST (EST) subfamilies. Amino acid sequence motifs exist within ST enzymes that are conserved throughout phylogeny. These signature sequences may be involved in the binding of 3'-phosphoadenosine-5 '-phosphosulfate, the cosubstrate for the sulfonation reaction. There are presently five known human cytosolic ST enzymes: an EST, an HSST, and three PSTs. cDNAs and genes for all of these enzymes have been cloned, and chromosomal localizations have been reported for all five genes. Genes for these human enzymes, as well as those of other mammalian cytosolic ST enzymes that have been cloned, show a high degree of structural homology, with conservation of the locations of most intron/exon splice junctions. Human ST enzyme expression varies among individuals. Functionally significant genetic polymorphisms for ST enzymes in humans have been reported, and other molecular genetic mechanisms that might be involved in the regulation of the expression of these enzymes are being explored. Knowledge of the molecular biology of cytosolic ST enzymes, when placed within a context provided by decades of biochemical research, promises to significantly enhance our understanding of the regulation of the sulfate conjugation of hormones, neurotransmitters, and drugs.

Animals↗

PubMed1996

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E.coli rho protein.

Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1-116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)10 to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1-130 of rho protein (Rho130) is found to be stably folded, both in absence and presence of nucleic acid. NMR studies of the complex of Rho130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho130 are similar to those of intact rho protein; therefore, Rho130 is a suitable model of the RNA-binding domain of Rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggests that Rho130 forms oligomers in the presence of longer oligonucleotides. 1H, 15N and 13C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.

Amino Acid Sequence↗

PubMed1996

Human dehydroepiandrosterone sulfotransferase pharmacogenetics: quantitative Western analysis and gene sequence polymorphisms.

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of DHEA and other hydroxysteroids. DHEA ST enzymatic activity in individual human liver biopsy samples has been shown to vary over a five-fold range, and frequency distribution histograms are bimodal, with approximately 25% of subjects included in a high activity subgroup. We set out to characterize the molecular basis for variation in human liver DHEA ST activity. The first step involved performing quantitative Western analysis of cytosol preparations from 92 human liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. There was a highly significant correlation (r(s) = 0.635, P < 0.0001) between levels of DHEA ST activity and immunoreactive protein. We next attempted to determine whether the expression of DHEA ST might be controlled, in part, by a genetic polymorphism. DNA was isolated from three "low" and three "high" DHEA ST activity liver samples. Exons and the 5'-flanking region of the DHEA ST gene (STD) were amplified for each of these samples with the polymerase chain reaction (PCR). When compared with "wild type" STD sequence, some of the samples contained a T --> C transition at DHEA ST cDNA nucleotide 170, located within exon 2, resulting in a Met 57 --> Thr change in amino acid. Other samples contained an A --> T transversion at nucleotide 557 within STD exon 4 that resulted in a Glu 186 --> Val change. STD exons 2 and 4 were then sequenced for DNA isolated from an additional 87 liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. The allele frequency for the exon 2 polymorphism in these samples was 0.027, whereas that for the exon 4 polymorphism was 0.038, but neither polymorphism was systematically related to the level of enzyme activity in these samples. Transient expression in COS-1 cells of cDNA that contained the nucleotide 170 and 557 polymorphisms, either separately or together, resulted in decreased expression of both DHEA ST enzymatic activity and level of immunoreactive protein, but only when the nucleotide 557 variant was present. Identification of common genetic polymorphisms within STD will now make it possible to test the hypothesis that those polymorphisms might alter in vivo expression and/or function of this important human steroid-metabolizing enzyme.

Amino Acid Sequence↗

PubMed1995

Influence of protein and hormone supplementation on in vitro maturation and fertilization of domestic cat eggs.

Immature eggs were recovered from freshly excised ovaries from domestic cats, and initially 931 eggs with a compact cumulus and uniform cytoplasm were cultured in 1 of 15 treatments. Eagle's minimum essential medium containing glutamine and pyruvate was supplemented with 5% (v/v) fetal calf serum (FCS), 4 mg bovine serum albumin (BSA) ml-1 or 2 mg polyvinyl alcohol ml-1 (PVA; non-protein control). Within each of these supplement groups, eggs were cultured with: no hormone; LH+FSH; LH+FSH+oestradiol; or LH+FSH+oestradiol+progesterone. After incubation for 52 h, eggs were inseminated with conspecific fresh spermatozoa, cultured and examined for stage of meiotic maturation and in vitro fertilization (IVF). There were fewer (P < 0.05) eggs maturing to metaphase II in vitro in FCS compared with BSA or PVA, the last two treatments producing similar (P < 0.05) results. Gonadotrophins in concert with oestradiol or oestradiol+progesterone improved the incidence of maturation (P < or = 0.01) compared with no added hormones. The incidence of fertilization and cleavage in vitro ranged from 5.2 to 33.9% and varied (P < 0.05) with hormone subtreatment. Adding FSH+LH+oestradiol consistently increased the incidence of IVF approximately twofold compared with controls with no added hormones. Although it inhibited the ability of eggs to reach metaphase II, FCS in the presence of gonadotrophins and oestradiol allowed > 60% of mature eggs to fertilize in vitro (P < 0.05, compared with PVA and BSA). Inhibitory effects on egg maturation were further evaluated by testing four FCS batches from three commercial sources against BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Human liver estrogen sulfotransferase: identification by cDNA cloning and expression.

Sulfation is an important pathway in the biotransformation of steroid hormones such as estrogens. Human liver contains three well-characterized sulfotransferase (ST) enzymes, dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Although two of these enzymes, DHEA ST and one form of PST, can catalyze the sulfate conjugation of estrogens, our goal was to test the hypothesis that human liver might also contain a distinct estrogen ST (EST). We used the PCR to clone a human liver EST cDNA by utilizing degenerate oligonucleotide primers designed on the basis of highly conserved EST sequences in non-human species to amplify a 512 nucleotide portion of the open reading frame (ORF) with single-stranded human liver cDNA as a template. Rapid amplification of cDNA ends (RACE) was then used to obtain the 5'- and 3'-ends of the EST cDNA. The ORF of this cDNA consisted of 882 nucleotides and encoded 294 amino acids. The protein encoded by the human liver EST cDNA was 81%, 73%, and 72% identical to the amino acid sequences of guinea pig adrenocortical, bovine placental and rat liver ESTs, respectively. Human liver EST transiently expressed in COS-1 cells was capable of catalyzing the sulfation of estrone, DHEA and 4-nitrophenol, but not that of dopamine. The expressed human liver EST was also characterized with regard to thermal stability, inhibition by 2,6-dichloro-4-nitrophenol (DCNP) and response to NaCl. Identification of human liver EST by cloning and expression of its cDNA should enhance understanding of the enzymology and regulation of the biotransformation of estrogens and other steroids in humans.

Amino Acid Sequence↗

PubMed1994

Human liver thermolabile phenol sulfotransferase: cDNA cloning, expression and characterization.

The sulfate conjugation of phenolic biogenic amines in humans is catalyzed by the thermolabile (TL) form of phenol sulfotransferase (PST). As a first step toward cloning a cDNA for TL PST, the enzyme was purified from jejunal mucosa, the human tissue with the highest known specific activity, and purified TL PST was subjected to limited proteolysis and amino acid sequencing. The PCR was then performed with human liver cDNA as template and primers designed on the basis of an 18 amino acid stretch of TL PST sequence to obtain a probe for screening cDNA libraries. When cDNA library screening proved unsuccessful, PCR primers were designed based on the nucleotide sequence of a functionally uncharacterized human brain cDNA that had been speculated to represent an aryl sulfotransferase. This brain cDNA encoded the 18 amino acid sequence that we had determined in TL PST. With human liver cDNA as template, these primers amplified a PCR product that contained an 885 nucleotide open reading frame that encoded 295 amino acids--including the 18 amino acids of TL PST sequence. In vitro transcription and translation of this human liver cDNA resulted in the synthesis of a 35.5 kDa protein. The human liver cDNA was also expressed in COS-1 cells, and the biochemical and physical characteristics of the encoded enzyme were identical with those of human liver TL PST. The cloning of a cDNA for human liver TL PST represents an important step toward understanding the molecular basis for the regulation of this enzyme in humans.

Amino Acid Sequence↗

PubMed1994

Human histamine N-methyltransferase pharmacogenetics: cloning and expression of kidney cDNA.

Histamine N-methyltransferase (HNMT) catalyzes the NT-methylation of histamine. The level of HNMT activity in human red blood cells is controlled by a common genetic polymorphism. We set out to clone and express a cDNA for HNMT from human tissue as a first step toward a determination of the molecular basis for this genetic polymorphism. The cloning strategy was based on possible sequence homology between rat and human kidney HNMT. Human kidney cDNA libraries were screened with the 885-nucleotide open reading frame of rat kidney HNMT cDNA. A 1.4-kilobase cDNA clone was isolated that contained two potential translation initiation codons, both in the same reading frame. The longest open reading frame of the human kidney cDNA clone contained 876 nucleotides and encoded a protein 292 amino acids in length. The amino acid sequence of this protein was 84% identical to that of rat kidney HNMT. The human kidney cDNA clone was transcribed in vitro and translated in a rabbit reticulocyte lystate system to yield a protein with an apparent molecular mass of 33 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human kidney cDNA was also subcloned into the eukaryotic expression vector p91023(B). Partially purified HNMT isolated from the cytosol of GOS-1 cells transfected with this expression construct had biochemical properties similar to those of human kidney HNMT. Human renal cortical HNMT, partially purified human renal cortical HNMT, and partially purified transfected COS-1 cell HNMT had Km values for histamine and S-adenosyl-L-methionine, the two cosubstrates for the enzyme reaction, of 20, 13, and 14 microM and 2.0, 3.0, and 6.2 microM, respectively. IC50 values for the HNMT inhibitor amodiaquine were 0.50, 0.48, and 0.40 microM, respectively, for enzyme from these same three sources. Northern blot analyses performed with poly(A)+ RNA from a series of human tissues including kidney demonstrated three transcripts, approximately 1.3, 3.8, and 4.0 kilobases in length. Cloning of a cDNA for HNMT may now make it possible to determine the molecular basis for the HNMT genetic polymorphism in humans.

Amino Acid Sequence↗

PubMed1993

Circannual inter-relationships among reproductive hormones, gross morphometry, behaviour, ejaculate characteristics and testicular histology in Eld's deer stags (Cervus eldi thamin).

Blood samples, morphometric measurements and behavioural data were collected weekly for 52 weeks from six adult Eld's deer stags exposed to natural fluctuations in photoperiod (38 degrees N latitude). Mean (+/- SEM) serum LH concentrations reached peak values in the autumn (October), three months before FSH and testosterone concentrations reached peak values in early winter (January). Prolactin concentrations were inversely related (r = -0.733, P < 0.001) to LH and directly related to daylength, and maximal concentrations were observed during mid-summer (July) and minimal concentrations during early winter (January). The temporal pattern of circulating inhibin was positively correlated with FSH (r = 0.88, P < 0.001), but lagged behind the seasonal FSH increase by 1-3 weeks. Antler length, body weight and chest girth were maximal during pre-rut (December-January). Maximal scrotal circumference and combined testes volume were observed in mid-winter (February), whereas peak neck girth and behavioural aggression occurred 1-3 months later (March-May). On the basis of quarterly electroejaculation results, motile spermatozoa were produced in all seasons. However, the greatest number of motile spermatozoa per ejaculate was observed during the winter and spring, whereas the highest incidence of sperm pleiomorphisms (> 80%) was detected in the autumn. Histological assessments of the regressed testis (July) revealed fewer germ cells undergoing spermatogenesis and an increased incidence of degenerating and abnormal cell types. In summary, Eld's deer exhibit a circannual hypothalamic-pituitary-gonadal cycle with onset of pituitary activation occurring during the autumn and winter, whereas gonadal activity peaks during the winter and spring as daylengths are increasing. Marked circannual variations in circulating prolactin suggest that Eld's deer may use photoperiodic cues to modulate seasonal fertility; however, the existence of an endogenous seasonal rhythm operating independently of photoperiod cannot be excluded.

Animals↗

PubMed1993

Seasonal patterns of basal and GnRH-induced LH, FSH and testosterone secretion in Eld's deer stags (Cervus eldi thamin).

Plasma LH, FSH and testosterone were measured in blood samples collected via remote catheterization from six adult Eld's deer stags every 10 min for 8 h before and 2 h after GnRH (1 microgram kg-1, i.v.) administration. Blood samples were collected within 2 weeks of the summer solstice (21 June), autumn equinox (22 September), winter solstice (21 December) and spring equinox (20 March). Marked seasonal variations in basal LH, FSH and testosterone concentrations were observed. From autumn, well-defined LH pulses were temporally associated with small, but detectable pulses in testosterone. During the winter transition into the breeding season, episodic LH pulses were also temporally associated with corresponding testosterone surges that lasted 2-3 h. High amplitude, low frequency testosterone surges were also observed during the spring, but often in the absence of detectable LH pulses. Basal LH and testosterone concentrations decreased during the summer and, although LH pulses were detected, associated testosterone pulses were absent. Only 37% of LH pulses occurred coincidentally with FSH pulses, and FSH pulses were generally less prominent. The increases in LH and FSH above basal concentrations after GnRH treatment were significant (P < 0.05) for all seasons. Increases in testosterone after GnRH treatment were greatest during the winter and spring, but testosterone also increased to a lesser extent during the autumn (P < 0.05). In contrast, testosterone concentrations were not different before and after GnRH treatment during summer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Successful intrauterine insemination of Eld's deer (Cervus eldi thamin) with frozen-thawed spermatozoa.

This study tested the efficacy of assisted reproduction (synchronization of oestrus and intrauterine artificial insemination (AI)) in contributing to the captive propagation of an endangered species, the Eld's deer (Cervus eldi thamin). Semen was collected from males preselected on the basis of under-represented genotype. Motility of spermatozoa after thawing from ejaculates diluted with BF5F extender (8% glycerol), frozen on dry ice in 0.5 ml straws and stored in liquid nitrogen was 60-70%. Intravaginal progesterone-releasing devices (controlled internal drug release, CIDR-type G) were inserted into 20 adult Eld's deer hinds for 14 days. In all hinds, semen (7.5-10 x 10(6) motile spermatozoa per uterine horn) was deposited by laparoscopy performed 70 h after removal of the CIDR device. Ovarian activity, before and after AI, was monitored by analysing pregnanediol-3 alpha-glucuronide (PdG) concentrations in voided urine collected three to seven times per week. During the period of CIDR device insertion, urinary PdG profiles were equal to, or above, normal luteal phase concentrations in all hinds. Within 48 h of device withdrawal, PdG concentrations returned to baseline values in 17 of the 20 females, and the onset of behavioural oestrus occurred at this time in 12 hinds. On the basis of sustained increases in urinary PdG, 9 of the 20 hinds were diagnosed as pregnant by 90 days after AI, all of which delivered offspring after a mean gestation of 241.1 days (range, 235-245). Seven singletons (two females, five males) were born alive and survived, and one singleton and one set of twins were stillborn (three females).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Human thiopurine methyltransferase: molecular cloning and expression of T84 colon carcinoma cell cDNA.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. Levels of TPMT activity in human tissue are controlled by a common genetic polymorphism that is an important factor responsible for individual variation in thiopurine drug toxicity and therapeutic efficacy. Our goal was to purify, to obtain a partial amino acid sequence for, and to clone and express cDNA for human TPMT as a first step in determining the molecular basis for this genetic polymorphism. Human kidney TPMT was purified, the protein was subjected to limited proteolysis, and amino acid sequence information was obtained from the resultant peptide fragments. Primers based on the amino acid sequence information were used to amplify a unique sequence from human liver cDNA by use of the polymerase chain reaction. Because TPMT has been reported to be present in the colon, T84 human colon carcinoma cells were studied and were found to express TPMT activity with biochemical properties similar to those of the human kidney and liver enzymes. Oligonucleotide probes based on the human kidney TPMT amino acid sequence were then used to screen a T84 human colon carcinoma cell cDNA library. A 2.7-kilobase cDNA clone was isolated that contained an open reading frame of 735 nucleotides, which encoded a protein of 245 amino acids. The deduced amino acid sequence of the encoded protein included one 24- and two separate 12-amino acid sequences identical to those obtained by sequencing proteolytic fragments of purified human kidney TPMT. Transcripts were made in vitro from the open reading frame of the cDNA clone. These transcripts were translated in a rabbit reticulocyte lysate system, and the resulting translation product comigrated with human kidney TPMT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The T84 cell cDNA clone, truncated within the 3' untranslated region at an Sstl restriction site, was then used to create an expression construct with the eukaryotic expression vector P91023(B), and this construct was used to transfect COS-1 cells. The transfected cells expressed a high level of TPMT enzymatic activity, and this activity displayed a pattern of inhibition by TPMT inhibitors identical to that of human kidney and T84 human colon carcinoma cell TPMT. Cloning of cDNA for this important drug-metabolizing enzyme may make it possible to define the molecular basis of the TPMT genetic polymorphism in humans.

Amino Acid Sequence↗

PubMed1992

Human liver dehydroepiandrosterone sulfotransferase: molecular cloning and expression of cDNA.

Sulfation is an important pathway in the metabolism of many hormones and drugs. Human liver contains at least three well characterized sulfotransferase (ST) enzymes, i.e., dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Our goal was to purify, to obtain partial amino acid sequence for, and to clone and express cDNA for human liver DHEA ST. Polymerase chain reaction primers were designed on the basis of homology among rat liver hydroxysteroid ST, rat liver PST, and bovine estrogen ST. These primers amplified a unique sequence from human liver cDNA, and this polymerase chain reaction product was used to screen a human liver cDNA library. Two clones were isolated that contained identical open reading frames, of 855 nucleotides, that encoded a protein of 285 amino acids. The deduced amino acid sequence of the encoded protein included two separate 27- and 23-amino acid sequences that were identical to those obtained by microsequencing of proteolytic fragments from purified human liver DHEA ST. Translation, in a rabbit reticulocyte lysate system, of mRNA transcribed in vitro from the two cDNA clones resulted in a 35-kDa translation product that comigrated with purified human liver DHEA ST during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This translation product also catalyzed the sulfation of DHEA but not the sulfation of model substrates for the two forms of PST found in human liver. The two cDNA clones were also used to create expression constructs with the eukaryotic expression vector P91023(B), and these constructs were used to transfect COS-1 cells. The transfected cells expressed a high level of DHEA ST activity, and this enzyme activity displayed a pattern of inhibition by the ST inhibitor 2,6-dichloro-4-nitrophenol identical to that of human liver DHEA ST. Cloning of cDNA for this important human sulfate-conjugating enzyme will enhance understanding of the relationship between DHEA ST and other human liver STs, as well as ST enzymes in other species.

Amino Acid Sequence↗

PubMed1990

Formation and characterization of vesicles from day-10 horse conceptuses.

The objectives of this research were 1) to determine if cellular vesicles could be formed from Day-10 horse conceptuses similar to trophoblastic vesicles reported for other domestic species and 2) to characterize various aspects of their development in vitro and their ability to withstand the freeze-thaw process. Twenty-five conceptuses were recovered from lighthorse mares on Day 10 after ovulation. After two washes in 0.05% trypsin, each conceptus was placed in 0.25% trypsin until the capsule thinned. Mechanical dispersion with a glass pipette resulted in a combination of individual cells and cell clumps. When cultured in vitro, all preparations exhibited both partially and completely formed vesicles within 24 h. Cellular monolayers also developed within the first 24 h of culture and were predominant by 96 h. Within 13 to 20 d, all monolayers developed dense areas of cells that eventually released from the cell matrix and aggregated to form vesicles. Progesterone and total estrogen concentrations in media samples were lower (P < 0.05) for conceptuses that had required long trypsinization periods for dispersion. Pregnant mare serum gonadotropin was not detectable in any samples. Vesicles cultured in nontreated tissue culture flasks.doubled in size within 24 h but did not increase further by 48 h. Of 234 vesicles frozen after 48 h of culture, the postthaw viability, as measured by the ability to return to prefreeze characteristics, was 34, 21 and 11% after 24, 96 and 168 h in culture, respectively. We conclude that vesicles can be formed by enzymatic dispersion of Day-10 horse conceptuses. However, monolayers were the predominant result of both the initial dispersions and the long-term culture of vesicles. Vesicles showed a limited ability to grow in vitro.

Journal Article↗

PubMed1990

New trends in the management of peritonsillar abscess.

Peritonsillar abscess is the most common complication of acute tonsillitis. Signs and symptoms include fever, unilateral sore throat, odynophagia and trismus. Optimal management consists of antibiotic therapy and drainage of the abscess. Controversy exists about the drainage procedure, which includes needle aspiration, incision and drainage, or acute tonsillectomy. Data indicate that outpatient needle aspiration, antibiotics and pain medication are effective treatment in 85 to 90 percent of patients with uncomplicated peritonsillar abscesses.

Anti-Bacterial Agents↗