Student-selected components: bringing more ENT into the undergraduate curriculum.

Exposure to otolaryngology is currently minimal in the UK undergraduate medical curriculum. This may lead to difficulties in attracting graduates into higher ENT surgical training and in ensuring a reasonable standard of ENT knowledge amongst primary care practitioners. A recent innovation, of which many ENT units may be unaware, is the introduction to the undergraduate curriculum of 'student-selected components'. Like the traditional elective, this allows students to undertake an attachment to a speciality and department of their choice. Units which do not regularly teach medical students but which have a welcoming and enthusiastic approach to undergraduate training may well be ideal hosts. This paper introduces the concepts underlying student-selected components, outlines the preparation required and offers a template for such an attachment, for which ENT is ideally suited.

Identification of genes of Aspergillus fumigatus up-regulated during growth on endothelial cells.

Aspergillus fumigatus is an important opportunistic fungal pathogen that can cause acute invasive disease in neutropenic hosts. Invasive aspergillosis is being diagnosed with increasing frequency, and morbidity and mortality remain high despite prompt antifungal therapy. Because little is known about the virulence factors used by A. fumigatus, a tissue culture model was developed to mimic the interaction of the fungus with the endothelium. Differential display was used to compare gene expression in fungal cells grown on endothelial cells with that of cells grown in the absence of endothelial cell contact, and genes that were up-regulated were selected for analysis as putatively virulence-related genes. Two of these up-regulated genes were chosen for further study and were identified as genes encoding the regulatory subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase and a member of the ras gene family, both of which are involved in cAMP-mediated signaling in fungi. This model system provides a new approach to the identification of potentially virulence-related genes induced in A. fumigatus by the interaction with host cells.

Molecular cloning of cgrA, the gene encoding the Aspergillus nidulans ortholog of Saccharomyces cerevisiae CGR1.

Saccharomyces cerevisiae CGR1 encodes a 120-amino acid protein with a predominant nucleolar localization. In this study we report the identification and cloning of the ortholog, cgrA, from Aspergillus nidulans. The cgrA gene is comprised of three exons on A. nidulans Chromosome 7. The cDNA contains a single open reading frame (ORF) that would encode a protein of 114 amino acids with 44% sequence identity to yeast Cgr1p. A plasmid expressing cgrA complemented the impaired growth phenotype of a yeast strain that can be inducibly depleted of CGR1, and a green fluorescent protein (GFP)-tagged CgrA protein had the same nucleolar localization as the corresponding yeast protein. These results identify cgrA as the A. nidulans ortholog of yeast CGR1 and suggest evolutionary conservation of nucleolar localization mechanisms used by these proteins.

Cgr1p, a novel nucleolar protein encoded by Saccharomyces cerevisiae orf YGL0292w.

Saccharomyces cerevisiae open reading frame (ORF) YGL029w (CGR1) encodes a small hydrophilic protein of unknown function. To investigate the role of this gene, we have determined the intracellular localization of the encoded product and examined the effects of Cgr1p depletion on cell growth. Tagging Cgr1p with the green fluorescent protein (GFP) or the myc epitope showed focal accumulation of the fusion protein in the yeast nucleolus, and this localization overlapped with the distribution of the nucleolar protein Nop1p. Cells depleted of CGR1 mRNA were growth impaired and hypersensitive to the translational inhibitor paromomycin, and this phenotype was complemented by episomal expression of the CGR1-GFP fusion gene. These results identify Cgr1p as a novel component of the yeast nucleolus and suggest a potential role in ribosome biogenesis.

Molecular cloning of Aspergillus fumigatus CgrA, the ortholog of a conserved fungal nucleolar protein.

In this report we describe the cloning of cgrA, the Aspergillus fumigatus ortholog of the yeast nucleolar protein Cgr1p. The cgrA complementary DNA (cDNA) contains a single open reading frame that would encode a protein of 114 amino acids that has 42% sequence identity to yeast Cgrlp. Heterologous expression of a green fluorescent protein (GFP)-tagged A. fumigatus cgrA gene demonstrated that the CgrA protein could localize to the yeast nucleolus. Moreover, the cgrA cDNA complemented the growth deficiency caused by inducible depletion of intracellular Cgr1p levels in yeast. These results support an orthologous relationship between the CgrA and Cgr1 proteins, and open the way for future studies into the potential value of nucleolar proteins as antifungal targets.

Patient satisfaction with results of hysterectomy.

OBJECTIVE: The objectives of this study were to measure patient satisfaction with the results of hysterectomy and to determine factors associated with dissatisfaction. STUDY DESIGN: A total of 1299 women who underwent hysterectomy at 28 hospitals in Maryland were interviewed before and at 3, 6, 12, 18, and 24 months after the operation. RESULTS: At 12 and 24 months after the hysterectomy 95.8% and 96.0%, respectively, reported that the hysterectomy had completely or mostly resolved the problems or symptoms they had before surgery; 93.3% and 93.7%, respectively, reported that the results were better than or about what they expected; 85.3% and 81. 6%, respectively, reported that their health was better than before the hysterectomy; and 87.9% and 93.1%, respectively, reported being totally recovered. The factor most strongly and consistently associated with patient reports of negative outcomes was readmission because of a postdischarge complication. CONCLUSION: Postdischarge complication necessitating readmission plays an important role in patient dissatisfaction with the results of hysterectomy.

Effectiveness of hysterectomy.

OBJECTIVE: To measure the effectiveness of hysterectomy in relieving adverse symptoms and to identify factors associated with lack of symptom relief. METHODS: In a 2-year prospective study, data were collected before and at 3, 6, 12, 18, and 24 months after hysterectomy in 1,299 women who had hysterectomies for benign conditions at 28 hospitals across Maryland. Effectiveness was measured in terms of relief of symptoms such as problematic vaginal bleeding, pelvic pain, and urinary incontinence. Psychologic function and quality of life before and after surgery also were assessed. RESULTS: Symptom severity, depression, and anxiety levels decreased significantly after hysterectomy and quality of life improved, particularly in the area of social function. However, 8% of women had at least as many symptoms at problematic-severe levels 1 and 2 years after hysterectomy as before. In multiple logistic regression, several presurgical patient characteristics predicted lack of symptom relief, including therapy for emotional or psychologic problems, depression, and household income of $35,000 or less. Bilateral oophorectomy predicted lack of symptom relief at 24 months but not at 12 months after hysterectomy. CONCLUSION: Significant improvements were seen after hysterectomy for all three aspects of health status (symptoms, psychologic function, and quality of life), which persisted or continued to improve throughout the 2 years of follow-up. However, hysterectomy did not relieve symptoms for some women, particularly those who had low incomes or were in therapy at the time of hysterectomy.

Ovarian volume and menopausal status.

OBJECTIVE: The purposes of this study were to (1) examine whether ovarian volume differs by age and menopausal status in healthy women; (2) evaluate whether ovarian volume could be a sensitive and specific predictor of menopausal status; and (3) assess whether ovarian volume is affected by cigarette smoke, oral contraceptives (OCs), and hormone replacement therapy (HRT). DESIGN: Each participant (527 women) completed an extensive in-home interview that assessed age, menopausal status, smoking history, OC use, and HRT use. Each participant also received a transvaginal ultrasound that measured ovarian volume. Geometric means for ovarian volume were compared between premenopausal and postmenopausal women using t tests. Tests for trends were conducted using linear regression analyses. RESULTS: Ovarian volume declined with age (p < or = 0.0001) and also differed by menopausal status; postmenopausal women had smaller ovarian volumes than premenopausal women of the same age (p < or = 0.0001). Ovarian volume was not associated with smoking history or HRT use. However, it was significantly smaller in current users of OCs compared with past users of or those who never used OCs (p < or = 0.0001). Ovarian volume was a sensitive and specific predictor of postmenopausal status. CONCLUSIONS: The data suggest that age, menopausal status, and OC use may be determinants of ovarian volume. They also suggest that ovarian volume may be useful for predicting menopausal status in women.

Hysterectomy and sexual functioning.

CONTEXT: Women considering hysterectomy often are concerned about its potential effects on their sexual functioning but the effects of hysterectomy on sexual functioning remain unclear. OBJECTIVE: To examine changes in sexual functioning after hysterectomy. DESIGN AND SETTING: A 2-year prospective study (Maryland Women's Health Study) of hysterectomy, which included measures of sexual functioning prior to hysterectomy and at 6, 12, 18, and 24 months after hysterectomy, performed during 1992 and 1993. PATIENTS: Of 1299 women interviewed prior to hysterectomy, 1101 (84.8%) completed the study and provided information about their sexual functioning. Most were between the ages of 35 and 49 years, white, married or living with a partner, and high school graduates. MAIN OUTCOME MEASURES: Frequency of sexual relations, dyspareunia, orgasm, vaginal dryness, and sexual desire. RESULTS: The percentage of women who engaged in sexual relations increased significantly from 70.5% before hysterectomy to 77.6% and 76.7% at 12 and 24 months after hysterectomy. The rate of frequent dyspareunia dropped significantly from 18.6% before hysterectomy to 4.3 % and 3.6% at 12 and 24 months after hysterectomy. The rates of not experiencing orgasms dropped significantly from 7.6% before hysterectomy to 5.2% and 4.9% at 12 and 24 months after hysterectomy. Low libido rates also decreased significantly from 10.4% before hysterectomy to 6.3% and 6.2% at 12 and 24 months after hysterectomy. The distribution of women not reporting vaginal dryness in the past month improved significantly from 37.3% before hysterectomy to 46.8% and 46.7% at 12 and 24 months after hysterectomy. Prehysterectomy depression was associated with experiencing dyspareunia, vaginal dryness, low libido, and not experiencing orgasms after hysterectomy. CONCLUSIONS: Sexual functioning improved overall after hysterectomy. The frequency of sexual activity increased and problems with sexual functioning decreased.

Sequencing of a gene encoding a member of the mitochondrial carrier family of transport proteins from Aspergillus nidulans.

Mitochondrial carrier proteins comprise a superfamily of evolutionarily conserved proteins that regulate the specific transport of essential metabolites across the mitochondrial membranes. In this report we describe the cloning and sequencing of a gene from Aspergillus nidulans, amc-1, that encodes the first reported example of a mitochondrial carrier protein in Aspergillus species. The amc-1 gene is located on chromosome 7, and is transcribed as a 1.6 kb unspliced polyadenylated RNA. The predicted translation product of the amc-1. cDNA displays three tandemly repeated domains which possess protein signature motifs that are characteristic of mitochondrial carrier proteins that localize to the inner mitochondrial membrane. amc-1 shares the greatest similarity with a Neurospora mitochondrial carrier protein that is implicated in basic amino acid transport, suggesting that the amc-1 protein may provide a related function.

Utility of routine testing of bronchoalveolar lavage fluid for cryptococcal antigen.

All cryptococcal antigen (CrAg) testing performed at our institution between 1989 and 1994 was reviewed for utility of routinely testing of bronchoalveolar lavage fluid (BAL) for this antigen. Forty-two of 1,506 BAL specimens were positive. Seventeen of these were felt to represent false positives (sensitivity, 71%; positive predictive value, 0.59). The data on CrAg in cerebrospinal fluid and serum and the fungal culture and histological results of BAL specimens did not support continued, routine testing of BALs for CrAg to diagnose cryptococcosis.

Evolutionary conservation of putative functional domains in the human homolog of the murine His-1 gene.

The mouse His-1 gene encodes a spliced and polyadenylated RNA with no long open reading frame (ORF), making it difficult to distinguish a functional protein coding domain. To identify candidate protein coding ORFs, and other functionally significant regions, we have isolated and sequenced 8.5 kb of a human genomic DNA that is homologous to the mouse His-1 gene. Alignment of the mouse and human sequences required no extensive gapping, indicating that evolutionary constraints have maintained a requirement for colinearity in genomic organization. We have identified the mouse transcriptional start point (tsp) and shown that the sequence of the 5'-flanking region is highly conserved in the human homolog. Sequence comparisons between the mouse and human genes identified conservation of other putative functional domains in exon 3 and in each of the two introns. Southern blot analysis with probes from each of these regions detected homologs in multiple other vertebrate species. However, none of the multiple candidate ORFs in the mouse RNA were conserved in the human sequence, suggesting that the RNA is unlikely to encode a protein. These data suggest that the RNA may be the final and functional product from the mouse His-1 gene.

Characterization of a putative retroviral env-related human protein.

In this study, we developed a rabbit polyclonal antibody to a fusion protein containing the envelope (env) transmembrane (TM) region from the human endogenous retroviral family, HERV-E. We used this reagent to document the expression of TM-related protein by Western blot in endothelial, colon and prostate carcinoma and seminoma cell lines and in peripheral blood mononuclear cells from a healthy donor. We detected a 58-kD protein (as compared to murine TM of 15 kD) that had specificity for the env-related antibodies of the polyclonal antiserum.

The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis.

Two chitin synthase genes, chsD and chsE, were identified from the filamentous ascomycete Aspergillus nidulans. In a region that is conserved among chitin synthases, the deduced amino acid sequences of chsD and chsE have greater sequence identity to the polypeptides encoded by the Saccharomyces cerevisiae CHS3 gene (also named CSD2, CAL1, DIT101, and KTI1) and the Candida albicans CHSE gene than to other chitin synthases. chsE is more closely related to the CHS3 genes, and this group constitutes the class IV chitin synthases. chsD differs sufficiently from the other classes of fungal chitin synthase genes to constitute a new class, class V. Each of the wild-type A. nidulans genes was replaced by a copy that had a substantial fraction of its coding region replaced by the A. nidulans argB gene. Hyphae from both chsD and chsE disruptants contain about 60-70% of the chitin content of wild-type hyphae. The morphology and development of chsE disruptants are indistinguishable from those of wild type. Nearly all of the conidia of chsD disruption strains swell excessively and lyse when germinated in low osmotic strength medium. Conidia that do not lyse produce hyphae that initially have normal morphology but subsequently lyse at subapical locations and show ballooned walls along their length. The lysis of germinating conidia and hyphae of chsD disruptants is prevented by the presence of osmotic stabilizers in the medium. Conidiophore vesicles from chsD disruption strains frequently swell excessively and lyse, resulting in colonies that show reduced conidiation. These properties indicate that chitin synthesized by the chsD-encoded isozyme contributes to the rigidity of the walls of germinating conidia, of the subapical region of hyphae, and of conidiophore vesicles, but is not necessary for normal morphology of these cells. The phenotypes of chsD and chsE disruptants indicate that the chitin synthesized by each isozyme serves a distinct function. The propensity of a chsD disruptant for osmotically induced lysis was compared to that of strains carrying two other mutations (tsE6 and orlA::trpC) which also result in reduced chitin content vegetative cell lysis. The concentration of osmotic stabilizer necessary to remedy the lysis of strains carrying the three mutations is inversely related to the chitin content of each strain. This finding directly demonstrates the importance of chitin to the integrity of the cell wall and indicates that agents that inhibit the chsD-encoded chitin synthase could be useful anti-Aspergillus drugs.

Expression of a putative immunosuppressive protein in human tumors and tissues.

Tumor-induced immunosuppression by murine retrovirus-induced tumors and nonviral murine and human tumors has been shown to be mediated by the transmembrane (TM) envelope (env) protein p15E. This in vitro activity is inhibitable by anti-(murine)p15E antibodies, implying that a TM-like protein is produced by such tumors. The leading candidate genes that might encode such proteins in human tumors are human endogenous retroviral (HERV) sequences. We have utilized immunohistochemistry to determine what tissues may express HERV env proteins. We subcloned a restriction fragment from the putative TM human env gene of a type C-related HERV (clone-4-1) into a fusion protein gene construct. Using a rabbit polyclonal antiserum against the fusion protein, we observed staining in a variety of human tumor and nontumor tissues.

Phaeohyphomycosis caused by Dactylaria (human dactylariosis): report of a case with review of the literature.

Phaeohyphomycosis due to Dactylaria (Ochroconis) spp. is a rare infection of man. It was first reported in 1986. All patients have had significant immunosuppression. To our knowledge, this is the second case of phaeohyphomycosis caused by Dactylaria constricta var. gallopava in a liver transplant patient and it developed even though he had been receiving fungal prophylaxis with fluconazole. Moreover, this case may represent nosocomial acquisition. In addition, we have reviewed the English language literature of previously reported patients with phaeohyphomycosis caused by Dactylaria spp.

Near-isogenic lines of maize differing for glycinebetaine.

A series of near-isogenic glycinebetaine-containing and -deficient F8 pairs of Zea mays L. (maize) lines were developed. The pairs of lines differ for alternative alleles of a single locus; the wild-type allele conferring glycinebetaine accumulation is designated Bet1 and the mutant (recessive) allele is designated bet1. The near-isogenic lines were used to investigate whether glycinebetaine deficiency affects the pool size of the glycinebetaine precursor, choline, using a new method for glycinebetaine and choline determination: stable isotope dilution plasma desorption mass spectrometry. Glycinebetaine deficiency in maize was associated with a significant expansion of the free choline pool, but the difference in choline pool size was not equal to the difference in glycinebetaine pool size, suggesting that choline must down-regulate its own synthesis. Consistent with this, glycinebetaine deficiency was also associated with the accumulation of the choline precursor, serine. A randomly amplified polymorphic DNA marker was identified that detects the bet1 allele. In 62 F8 families tested the 10-mer primer 5'-GTCCTCGTAG produced a 1.2-kb polymerase chain reaction product only when DNA from Bet1/bet1 or bet1/bet1 lines was used as template. All 26 homozygous Bet1/Bet1 F8 families tested were null for this marker.

Detection of cryptococcal antigen in bronchoalveolar lavage fluid: a prospective study of diagnostic utility.

Cryptococcal pneumonia is associated with significant morbidity and mortality in immunocompromised patients. We examined the utility of screening bronchoalveolar lavage (BAL) fluid for cryptococcal antigen. In a pilot study, we found that cryptococcal antigen was always positive in unprocessed BAL specimens of seven patients with cryptococcal pneumonia and negative in 44 patients with other granulomatous diseases who acted as the control subjects. A prospective study was done of 220 immunocompromised patients (188 with human immunodeficiency virus infection, 32 with other causes of immunosuppression) undergoing BAL for fever and pulmonary symptoms. The eventual diagnosis of cryptococcal pneumonia was made in eight patients. All eight patients had a cryptococcal antigen titer greater than or equal to 1:8. There were four patients without cryptococcal pneumonia who had cryptococcal antigen titers of 1:8, there were none with higher titers. For a titer of cryptococcal antigen titer of greater than or equal to 1:8, there was 100% sensitivity, 98% specificity, a positive predictive value of 67%, and a negative predictive value of 100%. The measurement of cryptococcal antigen in the BAL can be a rapid, simple way to make a diagnosis of cryptococcal pneumonia in immunosuppressed patients with pneumonia.

Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Delta-Pyrroline-5-Carboxylate Reductase.

Tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCl accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of Delta(1)-pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of Delta(1)-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCl-adapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and Delta(1)-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCl-dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties.