Biomedical subjects
K D Smith
Publications and source records attributed to K D Smith.
Transcripts homologous to a long repeated DNA element in the human genome.
Using three cloned DNA fragments from a 6.4-kb (kilobase pair) DNA element repeated several thousand times in the human genome (Adams, J. W., Kaufman, R. E., Kretschmer, P. J., Harrison, M. and Nienhuis, A. W. (1980) Nucleic Acids Res. 8, 6113-6128) and DNA/RNA hybridization, we show that transcripts homologous to this DNA family exist in total cellular RNA from human blood cells and from a mouse-human hybrid cell line with one human chromosome, the X. No such transcripts were detected in RNA from rabbit blood or a mouse cell line. For each DNA fragment studied, we found that blood transcripts and X-chromosome transcripts were indistinguishable in electrophoretic mobility and very heterogeneous in length; in addition, prominent hybridization bands were seen at 4.7 and 1.9 kb. Transcription from this DNA family likely occurs from heterogeneous templates. The existence of RNAs smaller than 6.4 kb suggests that part of the repeat unit can be transcribed and/or there exists a cellular mechanism to make these short RNAs from longer precursors. The vast majority of the RNAs homologous to the long repeat are not polyadenylated. In blood RNA there are a few hundred copies of beta-globin mRNA for every transcript homologous to this 6.4 kb repeat.
Acne and hyperandrogenism.
Elevated serum androgen levels have been reported in patients with acne resistant to conventional dermatologic therapy. This study was designed to investigate the relationship between serum androgen levels and the presence of acne in an unselected population of women seen consecutively by a dermatologist for various dermatologic complaints. Elevated serum testosterone levels were associated with acne regardless of whether this was the presenting complaint or an incidental finding. Women with both acne and hirsutism had higher serum testosterone levels than those with acne alone. Higher incidence of irregular menstrual cycles was noted in women complaining of acne. Normal serum testosterone levels were found only in those patients with regular menstrual cycles and the absence of acne or hirsutism. In conclusion, this study suggests that elevated serum testosterone levels are related to the presence of acne. Attention is called to the possibility that acne may be a clinical manifestation of a disorder with systemic and reproductive consequences.
Acne and hyperandrogenism: impact of lowering androgen levels with glucocorticoid treatment.
The impact of lowering androgen levels with glucocorticoid treatment in a group of consecutive female patients presenting to the Department of Reproductive Medicine and Biology, University of Texas, with a chief complaint of acne has been studied. One hundred fifty-eight patients, ages 16 to 40, who received prednisone at a maximum daily dose ranging from 7.5 to 15 mg for a period of at least 6 months were selected for the study. These patients were not taking other systemic medication affecting androgens or acne. Only thirty patients (19%) had pretreatment testosterone levels below the upper limits (40 ng/dl) of our normal range; while on treatment 146 patients (92.4%) had testosterone levels below 40 ng/dl. In sixty-three patients (39.9%) the acne completely cleared, in eighty patients (50.6%) it significantly improved, and in only fifteen patients (9.5%) was the acne not affected by the medication. There was a highly significant difference between the mean testosterone levels during treatment of those who cleared or improved, versus those who did not (p less than 0.05), the percentage drop in testosterone being greatest in those who cleared. Pretreatment testosterone levels were not significantly different in those who cleared, improved or did neither. It is concluded that glucocorticoid treatment frequently results in a lowering of androgen levels in hyperandrogenic women with acne and that this is associated with clearing or improvement of the acne.
Isolation of cDNA clones for rabbit red cell carbonic anhydrase and catalase: a pilot study directed at isolation of coordinately expressed genes.
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Diaphanography as a means of detecting breast cancer.
Diaphanography (lightscanning) is a noninvasive method of examining the breast by transillumination using visible or infrared light. It was compared to xeromammography in 1,000 patients, 91 of whom had biopsy. There were 34 malignant tumors, of which 29 were detected by xeromammography and 26 by lightscanning; 27 patients with positive lightscans were not biopsied. All patients with true-positive lightscans also had positive mammograms. Major criteria of malignancy on diaphanography included an area of light absorption and abnormal or hypervascularity. These criteria could be demonstrated on retrospect on 6 scans previously considered negative; one xeromammogram previously interpreted as negative was diagnosed as positive on retrospect. Diaphanography may be sensitive enough to warrant further investigation.
Testosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate in hyperandrogenic women.
Serum levels of testosterone (T), dehydroepiandrosterone (DHEA), and DHEA sulfate (DHEA-S) were measured in a group of 285 women with clinical signs of hyperandrogenism (hirsutism and/or acne). Levels of T were elevated in 75.8% of the patients, DHEA in 57.5%, and DHEA-S in 20%. Normal levels of all 3 androgens were found in 13.3%. Significant correlations were demonstrated among levels of all androgens. Only 7.7% of the patients had elevated levels of DHEA in the presence of normal T, and 3.2% had elevated DHEA-S and normal T levels. Subdivision of the study population on the basis of presence of acne, hirsutism, or both demonstrated no significant differences in androgen levels. Amenorrheic women had higher serum T levels than those with menstrual cycles. Women with laparoscopically demonstrated polycystic ovaries had significantly higher serum androgen levels than hyperandrogenic women with no laparoscopic evidence for polycystic ovarian disease. All 285 patients were treated with chronic low dose prednisone therapy. Overall suppression of all 3 androgens occurred in a large proportion of the patients. The pretreatment levels of DHEA or DHEA-S did not predict the responsiveness of T to chronic glucocorticoid therapy. On the other hand, a 2-day dexamethasone (DEX) suppression test quantitatively predicted the degree of T suppression achieved by chronic therapy. An overnight DEX suppression test was considerably less precise for this purpose. In conclusion, chronic low dose prednisone therapy resulted in suppression of serum T levels in a large proportion of women with hyperandrogenism of undetermined cause. The response to therapy could not be predicted on the basis of pretreatment serum DHEA or DHEA-S levels, but was predicted with a 2-day DEX suppression test.
Effect of acute and chronic androgen suppression by glucocorticoids on gonadotropin levels in hirsute women.
Hirsute women may have increased serum LH levels and increased ratios of LH to FSH in serum as well as increased serum androgen levels. Glucocorticoid therapy lowers serum testosterone (T) levels in some hirsute women, but no significant effects on gonadotropin levels have been reported. Sixty hirsute women had serum T, LH, and FSH levels measured before and after acute and chronic glucocorticoid administration. Both acute and chronic treatment resulted in significant suppression of T levels. Serum LH levels significantly decreased after chronic therapy. Significant direct correlations were found between T and LH or T and the LH to FSH ratio, both before and after therapy. In 11 women with normal T levels, acute or chronic glucocorticoid treatment did not produce a significant change in LH levels or LH to FSH ratios. Women (n = 26) with elevated T levels that were suppressed more than 50% during treatment had significant decreases in their mean LH levels and LH to FSH ratios. No significant change in mean LH or LH to FSH ratio occurred in women (n = 23) with elevated T levels that were not suppressed by 50%. These results demonstrate that chronic, but not acute, glucocorticoid-induced suppression of T levels in hyperandrogenic hirsute women results in lowering of LH and LH to FSH ratios.
Production of F cells in sickle cell anemia: regulation by a genetic locus or loci separate from the beta-globin gene cluster.
Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2% to 50% in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the beta-globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from beta S. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9% confidence limits or were alike by t test (P greater than or equal to .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always less than or equal to .005 and usually less than or equal to 10(-4). We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of beta-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the beta-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to beta S, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one.
Isolation and characterization of a major tandem repeat family from the human X chromosome.
We report the identification and characterization of a family of repeated restriction fragments whose molecular organization is apparently specific to the human X chromosome. This fragment, identified as an ethidium bromide-staining 2.0 kilobase (kb) band in BamHI-digested DNA from a Chinese hamster-human somatic cell hybrid containing a human X chromosome, has been cloned into pBR325 and characterized. The 2.0 kb repeated family has been assigned to the Xp11 leads to Xq12 region on the X by Southern blot analysis of somatic cell hybrids and is predominantly arranged in tandem clusters of up to seven 2.0 kb monomers. Homologous DNA sequences, not organized as 2.0 kb BamHI fragments, are found elsewhere on the X chromosome and on at least some autosomes, but are not found on the Y chromosome. From a dosing experiment using various amounts of the cloned repeat, we estimate that there are 5,000-7,500 copies of the 2.0 kb BamHI repeat per haploid genome. Since the vast majority, if not all, of these are confined to the X chromosome, this repeated DNA family must account for 5-10% of all X chromosome DNA and must constitute the major sequence component of the pericentromeric region of the X.
Adjuvants to immunological methods for mRNA purification. Application to the isolation of mRNA for carbonic anhydrase I from rabbit reticulocytes.
In order to generate the molecular probes needed to investigate the seemingly coordinate expression of carbonic anhydrase (CA I) and beta-globin within erythrocytes during human development, CA I-containing polyribosomes have been isolated from rabbit reticulocytes by reaction with purified antibodies to CA I followed by immunoadsorption of immune complexes with formalin-fixed protein A-bearing bacteria. In the course of such isolation, a series of maneuvers were seen to have a markedly favorable influence on the level of purity attained. These maneuvers include the use of 5 mg/ml of heparin concentrations to attenuate nonspecific binding and entrapment of unwanted polyribosomes, the addition of 200 units/ml of placental ribonuclease inhibitor to augment recovery in reactions which by test already appeared RNase-free, and the preadsorption of polyribosomes with formalin-fixed bacteria prior to immunological reaction so as to remove a subset of polyribosomes seemingly predisposed to nonspecific binding. In the absence of all of the maneuvers, attained purity was no greater than a few per cent. When all were employed, CA I-mRNA derived from immunopurified polyribosomes was recovered with more than 80% purity and 20% yield, as evident from both immunoassays and electrophoresis of its cell-free products.
Use of repetitive DNA for diagnosis of chromosomal rearrangements.
We have used two repeated DNA fragments (3.4 and 2.1 kb) released from Y chromosome DNA by digestion with the restriction endonuclease Hae III to analyze potential Y chromosome/autosome translocations. Two female patients were studied who each had an abnormal chromosome 22 with extra quinacrine fluorescent material on the short arm. The origin of the 22p+ chromosomes was uncertain after standard cytologic examinations. Analysis of one patient's DNA with the Y-specific repeated DNA probes revealed the presence of both the 3.4 and 2.1kb Y-specific fragments. Thus, in this patient, the additional material was from the Y chromosome. Analysis of the second patient's DNA for Y-specific repeated DNA was negative, indicating that the extra chromosomal segment was not from the long arm of the Y chromosome. These two cases demonstrate that repeated DNA can distinguish between similar appearing aberrant chromosomes and may be useful in karyotypic and prenatal diagnosis.
The relationship between the lengths of the follicular and luteal phases of the menstrual cycle and the fertility potential of the female.
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5-Azacytidine increases fetal hemoglobin production in a patient with sickle cell disease.
5-Azacytidine, given to one sickle cell patient as 9 doses of 30 mg/m2 each at intervals of 8 hr, rapidly increased the number of red cells containing HbF (F cells), decreased the amount of HbS in F cells, and decreased the MCHC of F cells. Although a significant rise in the peripheral blood hemoglobin from 8 gm/dl to 10-12 gm/dl was obtained, painful vaso-occlusive crises continued to occur. Therapy was associated with no marrow toxicity, although erythroblastosis appeared after each course of therapy. Increased HbF production was associated with demethylation of two restriction enzyme sites 5' to the two gamma-globin genes. Five other restriction sites around the gamma-globin genes were unchanged. 5-Azacytidine is considered potentially mutagenic and carcinogenic in man and, therefore, further experimental treatment should be limited only to severely affected sickle cell patients.
Evolution of human Y-chromosome DNA.
We have used human male-specific 3.4 kb Hae III restriction endonuclease fragments to explore the evolutionary history of man's Y-chromosome. We have identified four sets of reiterated sequences on the basis of their relative sequence homology with autosomal DNA. The sequences account for approximately 40% of the human Y-chromosome, are interspersed within the same 3.4 kb Hae III fragments, are heterogeneous and contain all reiterated DNA previously demonstrated to be specific for the Y-chromosome (it-Y DNA). Y-specific 3.4 kb Hae III sequences do not reassociate with either human female or ape DNA at standard reassociation criteria. However, approximately half of it-Y DNA (cross reacting it-Y) reassociates with both human female and ape DNA at reduced reassociation criteria. The remaining half (Y-specific it-Y) retains its specificity for the human Y-chromosome. These two sets of it-Y DNA have distinct reiteration frequencies and thermal stabilities with their Y-chromosome homologs. Non-Y-specific 3.4 kb Hae III sequences reassociate with both human female and ape DNA at standard reassociation criteria. The abundance of these non-Y-specific sequences decreases as a function of their evolutionary distance for man. One subset of non-Y-specific 3.4 kb Hae III sequences forms stable duplexes with human Y-chromosome DNA and with human and ape autosomal DNA. No detectable base-mismatch occurs among these homologs suggesting complete conservation of these sequences during primate evolution. The second subset of Non-Y-specific Hae III sequences form stable duplexes with human Y-chromosome DNA but highly mismatched duplexes with human and ape autosomal DNA. The finding that homologs of 3.4 kb Hae III sequences are not found within the Y-chromosome of apes but are only present in autosomes suggests that 3.4 kb Hae III sequences are largely autosomal in origin. Since autosomal homologs of most 3.4 kb Hae III-sequences exhibit a greater degree of divergence that those localized to the Y-chromosome, their evolutionary history seems to be chromosome-dependent. Our findings are not easily correlated with the comparative morphology of primate Y-chromosomes and suggest that sequence rearrangement has been a major event in the evolution of the human Y-chromosome. The significance of the specific interspersion of four sets of reiterated sequences, with distinct evolutionary histories, within a repeating unit specific to the human Y-chromosome is not clear. The apparent conservation of at least some of these reiterated sequences suggests they may be of functional importance.
The prognostic value of acute adrenal suppression and stimulation tests in hyperandrogenic women.
A group of 106 consecutively seen hyperandrogenic women were subjected to an acute adrenal suppression and stimulation test. The results of these tests were analyzed with respect to androgen suppression achieved after chronic glucocorticoid therapy in the same patients. The data suggested that an acute dexamethasone test may identify the group of hyperandrogenic women who respond poorly to chronic glucocorticoid therapy. This group of patients were found to have elevated luteinizing hormone (LH) levels and LH/follicle-stimulating hormone (FSH) ratios, suggesting the possibility of an LH-related hyperandrogenism. In patients whose elevated testosterone levels were suppressed by dexamethasone, adrenocorticotropic hormone (ACTH) induced a prompt return of the testosterone levels to baseline, suggesting an ACTH-dependent hyperandrogenism. In these patients, the degree of testosterone suppression after dexamethasone was not quantitatively related to the degree of testosterone suppression after chronic glucocorticoid therapy. In all cases chronic therapy resulted in a greater suppression of androgen levels than the acute dexamethasone test. In conclusion, an acute dexamethasone suppression test appears to be of clinical value in the management of the hyperandrogenic female, particularly in identification of women who will not respond to chronic glucocorticoid suppression therapy.
Cigarette smoking and semen quality.
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Response of the adrenal to adrenocorticotropic hormone (ACTH) in hyperandrogenic women treated chronically with low doses of prednisone.
A group of 43 women of reproductive age with clinical signs of hyperandrogenism was treated with a low dose (7.5 mg daily) of prednisone. An acute dexamethasone (DEX) suppression-adrenocorticotropic hormone (ACTH) stimulation test was performed before and after a minimum of 2 months of prednisone administration. The therapy produced a significant decrease of plasma testosterone (T) levels in a majority of patients. During the treatment period DEX failed to induce further suppression, while ACTH caused a significant rise in plasma T levels. The cortisol (F) levels were suppressed during treatment, but their response to ACTH stimulation was similar to that observed prior to initiation of therapy.