Improvement in scalp hair growth in androgen-deficient women treated with testosterone: a questionnaire study.

BACKGROUND: Androgens are thought to have an adverse effect on female scalp hair growth. However, our clinical experience of androgen replacement therapy in women with androgen deficiency, in which hair loss was seldom reported, led us to question this concept. OBJECTIVES: To evaluate the effect of subcutaneous testosterone therapy on scalp hair growth in female patients. METHODS: A total of 285 women, treated for a minimum of 1year with subcutaneous testosterone implants for symptoms of androgen deficiency, were asked to complete a survey that included questions on scalp and facial hair. Age, body mass index (BMI) and serum testosterone levels were examined. RESULTS: Out of the 285 patients, 76 (27%) reported hair thinning prior to treatment; 48 of these patients (63%) reported hair regrowth on testosterone therapy (responders). Nonresponders (i.e. no reported hair regrowth on therapy) had significantly higher BMIs than responders (P=0·05). Baseline serum testosterone levels were significantly lower in women reporting hair loss prior to therapy than in those who did not (P=0·0001). There was no significant difference in serum testosterone levels, measured 4weeks after testosterone implantation, between responders and nonresponders. No patient in this cohort reported scalp hair loss on testosterone therapy. A total of 262 women (92%) reported some increase in facial hair growth. CONCLUSIONS: Subcutaneous testosterone therapy was found to have a beneficial effect on scalp hair growth in female patients treated for symptoms of androgen deficiency. We propose this is due to an anabolic effect of testosterone on hair growth. The fact that no subject complained of hair loss as a result of treatment casts doubt on the presumed role of testosterone in driving female scalp hair loss. These results need to be confirmed by formal measurements of hair growth.

Multiple, compensatory regulatory elements specify spermatocyte-specific expression of the Drosophila melanogaster hsp26 gene.

The hsp26 gene of Drosophila melanogaster is expressed in six tissues during development and in a tissue-general response to heat shock. To be able to compare tissue-specific and heat-induced mechanisms of hsp26 expression, we have begun an analysis of the sequences involved in the spermatocyte-specific expression of the hsp26 gene by using germ line transformation. hsp26 mRNA synthesized in the spermatocytes has the same start site as sites previously demonstrated for nurse cell-specific and heat-induced mRNAs. Three regions of the hsp26 gene (nucleotides -351 to -135, -135 to -85, and +11 to +632) were able to stimulate spermatocyte-specific expression when fused with promoter sequences (nucleotides -85 to +11) that alone were insufficient to stimulate expression. These stimulatory regions appear to contain elements that provide redundant functions. While each region was able to stimulate expression independently, the deletion of any one region from a construct was without consequence as long as another compensatory region(s) was still present. There must reside, at a minimum, two independent spermatocyte-specifying elements within the sequences that encompass the three stimulatory regions and the promoter. At least one element is contained within sequences from -351 to -48. This region, in either orientation, can stimulate spermatocyte-specific expression from a heterologous promoter. A second element must reside in sequences from -52 to +632, since these sequences are also sufficient to direct spermatocyte-specific expression.

The structure of heterochromatic DNA is altered in polyploid cells of Drosophila melanogaster.

DNA sequences within heterochromatin are often selectively underrepresented during development of polyploid chromosomes, and DNA molecules of altered structure are predicted to form as a consequence of the underrepresentation process. We have identified heterochromatic DNAs of altered structure within sequences that are underrepresented in polyploid cells of Drosophila melanogaster. Specifically, restriction fragments that extend into centric heterochromatin of the minichromosome Dp(1;f)1187 are shortened in polyploid cells of both the ovary and salivary gland but not in the predominantly diploid cells of the embryo or larval imaginal discs and brains. Shortened DNA molecules were also identified within heterochromatic sequences of chromosome III. These results suggest that the structure of heterochromatic DNA is altered as a general consequence of polyploid chromosome formation and that the shortened molecules identified form as a consequence of heterochromatic underrepresentation. Finally, alteration of heterochromatic DNA structure on Dp(1;f)1187 was not correlated with changes in the variegated expression of the yellow gene located on the minichromosome.

Replication of heterochromatin and structure of polytene chromosomes.

Heterochromatin is characteristically the last portion of the genome to be replicated. In polytene cells, heterochromatic sequences are underreplicated because S phase ends before replication of heterochromatin is completed. Truncated heterochromatic DNAs have been identified in polytene cells of Drosophila and may be the discontinuous molecules that form between fully replicated euchromatic and underreplicated heterochromatic regions of the chromosome. In this report, we characterize the temporal pattern of heterochromatic DNA truncation during development of polytene cells. Underreplication occurred during the first polytene S phase, yet DNA truncation, which was found within heterochromatic sequences of all four Drosophila chromosomes, did not occur until the second polytene S phase. DNA truncation was correlated with underreplication, since increasing the replication of satellite sequences with the cycE(1672) mutation caused decreased production of truncated DNAs. Finally, truncation of heterochromatic DNAs was neither quantitatively nor qualitatively affected by modifiers of position effect variegation including the Y chromosome, Su(var)205(2), parental origin, or temperature. We propose that heterochromatic satellite sequences present a barrier to DNA replication and that replication forks that transiently stall at such barriers in late S phase of diploid cells are left unresolved in the shortened S phase of polytene cells. DNA truncation then occurs in the second polytene S phase, when new replication forks extend to the position of forks left unresolved in the first polytene S phase.

Sentinel lymph node biopsy for breast cancer: a suitable alternative to routine axillary dissection in multi-institutional practice when optimal technique is used.

PURPOSE: Previous studies have demonstrated the feasibility of sentinel lymph node (SLN) biopsy for nodal staging of patients with breast cancer. However, unacceptably high false-negative rates have been reported in several studies, raising doubt about the applicability of this technique in widespread surgical practice. Controversy persists regarding the optimal technique for correctly identifying the SLN. Some investigators advocate SLN biopsy using injection of a vital blue dye, others recommend radioactive colloid, and still others recommend the use of both agents together. PATIENTS AND METHODS: A total of 806 patients were enrolled by 99 surgeons. SLN biopsy was performed by single-agent (blue dye alone or radioactive colloid alone) or dual-agent injection at the discretion of the operating surgeon. All patients underwent attempted SLN biopsy followed by completion level I/II axillary lymph node dissection to determine the false-negative rate. RESULTS: There was no significant difference (86% v 90%) in the SLN identification rate among patients who underwent single- versus dual-agent injection. The false-negative rates were 11.8% and 5.8% for single- versus dual-agent injection, respectively (P <.05). Dual-agent injection resulted in a greater mean number of SLNs identified per patient (2. 1 v 1.5; P <.0001). The SLN identification rate was significantly less for patients older than 50 years as compared with that of younger patients (87.6% v 92.6%; P =.03). Upper-outer quadrant tumor location was associated with an increased likelihood of a false-negative result compared with all other locations (11.2% v 3. 9%; P <.05). CONCLUSION: In multi-institutional practice, SLN biopsy using dual-agent injection provides optimal sensitivity for detection of nodal metastases. The acceptable SLN identification and false-negative rates associated with the dual-agent injection technique indicate that this procedure is a suitable alternative to routine axillary dissection across a wide spectrum of surgical practice and hospital environments.

Optimal heat-induced expression of the Drosophila hsp26 gene requires a promoter sequence containing (CT)n.(GA)n repeats.

We report here the analysis of the sequence requirements for the heat-induced expression of the Drosophila melanogaster hsp26 gene using germline transformation. Heat-induced expression is augmented fivefold by a homopurine/homopyrimidine region from -85 to -134 that is devoid of heat-shock elements but contains numerous (dC-dT).(dG-dA) repeats. Sequences within this interval have been shown to assume a nuclease S1-hypersensitive structure in vitro. In this paper, we extend those in vitro observations, demonstrating that the S1-hypersensitive structure is triple-helical H-DNA formed by a symmetric (dC-dT).(dG-dA) sequence. Thus, the sequences that form H-DNA in vitro are also required in vivo for optimal hsp26 transcription. However, mutational analysis and diethylpyrocarbonate modification experiments in isolated nuclei suggest that the (dC-dT).(dG-dA) sequence does not form H-DNA in vivo and argue against a role for H-DNA in the heat-induced expression of hsp26.

Promoter sequence containing (CT)n.(GA)n repeats is critical for the formation of the DNase I hypersensitive sites in the Drosophila hsp26 gene.

We have analyzed P-element-transformed lines carrying hsp26/lacZ transgenes with various deletions and substitutions within the Drosophila melanogaster hsp26 promoter region in order to identify the sequences required for the formation of the DNase I hypersensitive sites (DH sites). DH sites are generally found associated with promoters and enhancer elements of active and inducible eukaryotic genes, and are thought to be nucleosome-free regions of DNA that interact with regulatory proteins and the transcriptional machinery. There are two major DH sites located within the promoter region of the hsp26 gene, centered at -50 and at -350 (relative to the hsp26 transcription start site). The sequences from -135 to -85, which contain (CT)n.(GA)n repeats, contribute significantly to the formation of the DH sites in the hsp26 promoter region. Deletion or substitution of this (CT)n region drastically reduces the accessibility of the DNA at these sites to DNase I. This reduction in accessibility was quantified by measuring the susceptibility of the DNA within nuclei to cleavage at a restriction site within the DH site. In addition to the (CT)n region and the promoter at -85 to +11 (region P), one of two other regions must be present for effective creation of the DH sites: sequences between -351 and -135 (region A), or sequences between +11 and +632 (region D). Disruption of the wild-type chromatin structure, as assayed by the loss of accessibility to the DH sites, is correlated with a decrease in inducible transcriptional activity, even when the TATA box and heat shock regulatory elements are present in their normal positions.

Practical guidelines for optimal gamma probe detection of sentinel lymph nodes in breast cancer: results of a multi-institutional study. For the University of Louisville Breast Cancer Study Group.

INTRODUCTION: Multiple radioactive lymph nodes are often removed during the course of sentinel lymph node (SLN) biopsy for breast cancer when both blue dye and radioactive colloid injection are used. Some of the less radioactive lymph nodes are second echelon nodes, not true SLNs. The purpose of this analysis was to determine whether harvesting these less radioactive nodes, in addition to the "hottest" SLNs, reduces the false-negative rate. METHODS: Patients were enrolled in this multicenter (121 surgeons) prospective, institutional review board-approved study after informed consent was obtained. Patients with clinical stage T1-2, N0, M0 invasive breast cancer were eligible. This analysis includes all patients who underwent axillary SLN biopsy with the use of an injection of both isosulfan blue dye and radioactive colloid. The protocol specified that all blue nodes and all nodes with 10% or more of the ex vivo count of the hottest node should be removed and designated SLNs. All patients underwent completion level I/II axillary dissection. RESULTS: SLNs were identified in 672 of 758 patients (89%). Of the patients with SLNs identified, 403 patients (60%) had more than 1 SLN removed (mean, 1.96 SLN/patient) and 207 patients (31%) had nodal metastases. The use of filtered or unfiltered technetium sulfur colloid had no impact on the number of SLNs identified. Overall, 33% of histologically positive SLNs had no evidence of blue dye staining. Of those patients with multiple SLNs removed, histologically positive SLNs were found in 130 patients. In 15 of these 130 patients (11.5%), the hottest SLN was negative when a less radioactive node was positive for tumor. If only the hottest node had been removed, the false-negative rate would have been 13.0% versus 5.8% when all nodes with 10% or more of the ex vivo count of the hottest node were removed (P =.01). CONCLUSIONS: These data support the policy that all blue nodes and all nodes with 10% or more of the ex vivo count of the hottest SLN should be harvested for optimal nodal staging.

Arteriovenous fistulas after cardiac catheterization.

In a review of five Dayton, Ohio, area hospitals during a six-year period, seven patients who were treated for an acquired arteriovenous (A-V) fistula after cardiac catheterization were identified. Four patients had undergone cardiac studies in area hospitals, while three were studied elsewhere. The four A-V fistulas after 23,291 cardiac catheterization procedures in Dayton hospitals represented an incidence of 0.017% for this complication. Congestive heart failure and limb ischemia were the most frequent presenting symptoms that developed from two to ten months after catheterization. Intentional puncture of both the artery and vein of the ipsilateral groin for right- and left-sided heart studies was the probable cause of fistula formation in two cases. Five patients sustained inadvertent injury to both an artery and adjacent vein during percutaneous vascular access. Six A-V fistulas that involved femoral vessels were managed by division of the fistula with lateral repair of the artery and vein. An unusual communication between the right thyrocervical trunk and the internal jugular vein was handled by ligation of the affected vessels. Prompt surgical correction of this unusual complication of percutaneous vascular access is recommended as spontaneous closure is unlikely.

Accuracy of sentinel lymph node biopsy for patients with T2 and T3 breast cancers.

Although numerous studies have demonstrated that sentinel lymph node (SLN) biopsy can accurately determine the axillary nodal status for early breast cancer some studies have suggested that SLN biopsy may be less reliable for tumors >2 cm in size. This analysis was performed to determine whether tumor size affects the accuracy of SLN biopsy. The University of Louisville Breast Cancer Sentinel Lymph Node Study is a prospective multi-institutional study involving 226 surgeons. The study was approved by the Institutional Review Board of each institution, and informed consent was obtained from all patients. Patients with clinical stage T1-2 N0 breast cancer were eligible for the study. Some patients with T3 tumors were included because they were clinically staged as T2 but on final pathology were found to have tumors >5 cm. This analysis includes 2148 patients who were enrolled from August 1997 through October 2000. All patients underwent SLN biopsy using a combination of radioactive colloid and blue dye injection followed by completion Level I/II axillary dissection. Statistical comparison was performed by chi-square analysis. The SLN identification rate, false negative rate, and overall accuracy of SLN biopsy were not significantly different among tumor stages T1, T2, and T3. We conclude that SLN biopsy is no less accurate for T2-3 breast cancers compared with T1 tumors.

Local DNA underreplication correlates with accumulation of phosphorylated H2Av in the Drosophila melanogaster polytene chromosomes.

DNA in Drosophila melanogaster polytene chromosomes is known to be locally underreplicated in both pericentric and intercalary heterochromatin. When the SuUR gene is mutant, complete and partial suppression of underreplication are observed in intercalary and pericentric heterochromatin, respectively; in contrast, overexpression of SuUR results in stronger underreplication. Using antibodies against phosphorylated histone H2Av and flies with different levels of SuUR expression, we demonstrated a clear correlation between the extent of underreplication in specific chromosome regions and the accumulation of H2Av phosphorylated at S137 (gamma-H2AX) at the same sites. Phosphorylated H2Av is a well-established marker of DNA double-stranded breaks (DSB). Our data thus argue that DNA underreplication leads to DSBs and that DSBs accumulate as salivary gland cells progress throughout repeated endocycles. We speculate that ligation of free double-stranded DNA termini causes the formation of ectopic contacts between the underreplicated regions in heterochromatin.

Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm.

This study compares the relative effects of advancing male age on multiple genomic defects in human sperm [DNA fragmentation index (DFI), chromatin integrity, gene mutations, and numerical chromosomal abnormalities], characterizes the relationships among these defects and with semen quality, and estimates the incidence of susceptible individuals for a well characterized nonclinical nonsmoking group of 97 men (22-80 years). Adjusting for confounders, we found major associations between age and the frequencies of sperm with DFI and fibroblast growth factor receptor 3 gene (FGFR3) mutations associated with achondroplasia (P < 0.01) with no evidence for age thresholds. However, we found no associations between age and the frequencies of sperm with immature chromatin, aneuploidies/diploidies, FGFR2 mutations (Apert syndrome), or sex ratio in this cohort. There were also no consistent correlations among genomic and semen-quality endpoints, except between DFI and sperm motility (r = -0.65, P < 0.001). These findings suggest there are multiple spermatogenic targets for genomically defective sperm with substantially variable susceptibilities to age. Our findings predict that as healthy males age, they have decreased pregnancy success with trends beginning in their early reproductive years, increased risk for producing offspring with achondroplasia mutations, and risk of fathering offspring with Apert syndrome that may vary across cohorts, but with no increased risk for fathering aneuploid offspring (Down, Klinefelter, Turner, triple X, and XYY syndromes) or triploid embryos. Our findings also suggest that the burden of genomic damage in sperm cannot be inferred from semen quality, and that a small fraction of men are at increased risk for transmitting multiple genetic and chromosomal defects.

Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis.

Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect.

Unusual properties of genomic DNA molecules spanning the euchromatic-heterochromatic junction of a Drosophila minichromosome.

While investigating the copy number of minichromosome Dp(1;f)1187 sequences in the polyploid chromosomes of ovarian nurse and follicle cells of Drosophila melanogaster we discovered that restriction fragments spanning the euchromatic-heterochromatic junction of the chromosome and extending into peri-centromeric sequences had the unusual property of being selectively resistant to transfer out of agarose gels during Southern blotting, leading to systematic reductions in Dp1187-specific hybridization signals. This property originated from the peri-centromeric sequences contained on the junction fragments and was persistently associated with Dp1187 DNA, despite attempts to ameliorate the effect by altering experimental protocols. Transfer inhibition was unlikely to be caused by an inherent physical property of repetitive DNA sequences since, in contrast to genomic DNA, cloned restriction fragments spanning the euchromatic-heterochromatic junction and containing repetitive sequences transferred normally. Finally, the degree of inhibition could be suppressed by the addition of a Y chromosome to the genotype. On the basis of these observations and the fact that peri-centromeric regions of most eukaryotic chromosomes are associated with cytologically and genetically defined heterochromatin, we propose that peri-centromeric sequences of Dp1187 that are incorporated into heterochromatin in vivo retain some component of heterochromatic structure during DNA isolation, perhaps a tightly bound protein or DNA modification, which subsequently causes the unorthodox properties observed in vitro.

Spatial and temporal pattern of hsp26 expression during normal development.

The tissue-specific patterns of developmental expression of hsp26-lacZ fusion genes inserted into Drosophila melanogaster by germline transformation were analyzed in several transformant lines utilizing a histochemical assay for beta-galactosidase activity on whole animals. We compared this pattern to the tissue-specific distribution of endogenous hsp26 RNA determined using hybridization of probes to RNA in situ in tissue sections. Both assays reveal that hsp26 is expressed in numerous tissues during development including spermatocytes, nurse cells, epithelium, imaginal discs, proventriculus and neurocytes. The ease and resolution of the whole-animal beta-galactosidase assay makes it particularly attractive for the elucidation of sequences involved in such complex regulation. The original hsp26-lacZ fusion gene contained 2 kb of sequence upstream of the transcription start. A construct containing only 278 bp upstream was still expressed in spermatocytes but no longer in nurse cells. In a few instances, the fusion genes were expressed in tissues for which there was no evidence for expression of the endogenous hsp26 gene. These novel patterns appear to be a result of chromosomal position since they were observed in only one or a subset of transformant lines containing identical inserts.

Replication forks are not found in a Drosophila minichromosome demonstrating a gradient of polytenization.

Differential DNA replication is widely held to influence polytene chromosome structure by causing the dramatic reductions in heterochromatic DNA content that are characteristic of most endopolyploid cells. The "underreplication model" of heterochromatic sequence underrepresentation predicts that replication intermediates should populate regions of DNA between fully polytenized euchromatic sequences and underpolytenized heterochromatic sequences. We directly tested this prediction using Dp1187, a 1300 kb Drosophila minichromosome containing well-defined heterochromatic regions. DNA from a euchromatic/heterochromatic junction region of Dp1187, demonstrating a significant gradient of underrepresentation in larval salivary glands, lacked the stalled replication forks predicted by the underreplication model. We consider an alternative mechanism leading to heterochromatic sequence underrepresentation involving a process of DNA elimination.

Effect of lodoxamide on in vitro and in vivo conjunctival immediate hypersensitivity responses in rats.

The antiallergic compound, lodoxamide, was evaluated for its abilities to attenuate a local allergic reaction in rat conjunctiva in vivo and to inhibit rat conjunctival mast cell mediator release in vitro. Topically applied lodoxamide (0.01, 0.10 and 1.0%, w/v) dose-dependently reduced the allergic response (23, 43 and 72%, respectively) in vivo. Lodoxamide was more effective than cromolyn sodium, N-acetyl aspartyl glutamic acid (Naaxia) and levocabastine, and 25 (7-200) times more potent than nedocromil sodium in direct comparisons. Addition of lodoxamide (10 micrograms/ml) to sensitized conjunctival tissue in vitro immediately prior to antigen challenge significantly reduced the amount of histamine released by the tissue. These data suggest that lodoxamide's in vivo anti-allergic activity in the conjunctiva is associated with its ability to prevent allergic mediator release from mast cells contained in this same tissue.

Determinants of heat shock-induced chromosome puffing.

Modified Drosophila heat shock genes were introduced into the germ line by P element transformation. The genes were altered such that several factors could be tested for their influence upon chromosome puffing. Deletion of promoter sequences upstream of position -73 of an hsp70-IacZ hybrid gene was sufficient to abolish puffing. Analysis of progressive 5' deletions defines a 16 bp interval that contains sequences required for both heat-induced puffing and gene expression. An internal deletion of the hsp70-IacZ gene that reduces the transcript size from 9 kb to 0.8 kb results in a dramatic reduction in puff size. The chromosomal insertion sites of 26 variant hsp70 or hsp26 genes fail to influence puffing greatly with one marked exception. This transformant possesses an insert that fails to puff and exhibits a tissue-restricted pattern of expression. These results indicate that variation in either promoter strength or transcript length have profound effects on puffing.

Histone H2A.Z is widely but nonrandomly distributed in chromosomes of Drosophila melanogaster.

Variant histones that differ in amino acid sequence from S-phase histones are widespread in eukaryotes, yet the structural changes they cause to nucleosomes and how those changes affect relevant cellular processes have not been determined. H2A.F/Z is a highly conserved family of H2A variants. H2Av, the H2A.F/Z variant of Drosophila melanogaster, was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation. H2Av was widely distributed in the genome and not limited to sites of active transcription. H2Av was present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes, and the H2Av antibody precipitated both transcribed and nontranscribed genes as well as noncoding euchromatic and heterochromatic sequences. The distribution of H2Av was not uniform. The complex banding pattern of H2Av in polytene chromosomes did not parallel the concentration of DNA, as did the pattern of immunofluorescence using H2A antibodies, and the density of H2Av measured by immunoprecipitation varied between different sequences. Of the sequences assayed, H2Av was least abundant on 1. 688 satellite sequences and most abundant on the hsp70 genes. Finally, transcription caused, to an equivalent extent, both H2Av and H2A to be less tightly associated with DNA.