Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps.

The levels of histone mRNA increase 35-fold as selectively detached mitotic CHO cells progress from mitosis through G1 and into S phase. Using an exogenous gene with a histone 3' end which is not sensitive to transcriptional or half-life regulation, we show that 3' processing is regulated as cells progress from G1 to S phase. The half-life of histone mRNA is similar in G1- and S-phase cells, as measured after inhibition of transcription by actinomycin D (dactinomycin) or indirectly after stabilization by the protein synthesis inhibitor cycloheximide. Taken together, these results suggest that the change in histone mRNA levels between G1- and S-phase cells must be due to an increase in the rate of biosynthesis, a combination of changes in transcription rate and processing efficiency. In G2 phase, there is a rapid 35-fold decrease in the histone mRNA concentration which our results suggest is due primarily to an altered stability of histone mRNA. These results are consistent with a model for cell cycle regulation of histone mRNA levels in which the effects on both RNA 3' processing and transcription, rather than alterations in mRNA stability, are the major mechanisms by which low histone mRNA levels are maintained during G1.

Metastatic carcinoma to the male breast.

A case of metastasis to the male breast from prostate carcinoma is reported. For proper treatment, it is important to differentiate primary from metastatic tumours. Prostate-specific antigen screening should be strongly considered in all breast masses seen in patients with known prostatic carcinoma. One cannot assume that breast enlargement in these patients is solely due to oestrogen-induced gynecomastia.

Metastatic melanoma of the tonsil and the nasopharynx.

Metastatic melanoma of the tonsils is rare. This paper describes one such case, highlighting the histopathology and the mode of spread.

Sleep nasendoscopy: what benefit to the management of snorers?

It has been proposed that sleep nasendoscopy (SN) will improve the success rate of the uvulopalatopharyngoplasty operation by identifying those patients with palatal snoring. The aim of this study was to test the efficacy of SN in the management of snorers who do not have obstructive sleep apnoea. This study compared a group of 26 snorers managed without SN (group A) to a group of 27 snorers managed with SN (group B). The post-operative results of group A were 61 per cent cured, 27 per cent better and 8 per cent unchanged. Group B results were 76 per cent cured, 19 per cent better and 5 per cent unchanged. However, if patients with only palatal snoring had surgery, the results for group B would have been 94 per cent cured, 6 per cent better and 0 per cent unchanged (95 per cent C.I. of difference +0.14, +0.54, p = 0.017). The results confirm the predictive power of SN in identifying success following uvulopalatopharyngoplasty. A simple grading system is suggested to aid in treatment planning. Patients are divided into three categories on SN: palatal snorers, mixed snorers and non-palatal (tongue base) snorers. It is proposed that uvulopalatopharyngoplasty may cure palatal snorers but mixed snorers will need additional therapies to eliminate their snoring although uvulopalatopharyngoplasty may improve the symptoms.

The evaluation of velopharyngeal function using flexible nasendoscopy.

Nasendoscopy is an essential tool in assessing the dynamic function and structure of the velopharyngeal sphincter during speech and swallowing. Flexible fibre-optic nasendoscopy has been used by the cleft palate team at Withington Hospital, Manchester since 1989. Seventy-six patients were referred between 1989 and 1994 for evaluation of velopharyngeal function during speech. Flexible nasendoscopic evaluation was attempted in 50 patients, and successfully carried out in 43 patients. The age range was four years to 77 years (mean 21 years). The patients were divided into two groups: Group 1 consisting of patients with cleft palate and Group 2 comprised of patients with non-overt cleft palate-related velopharyngeal dysfunction of various aetiologies; such as, submucous cleft, post-tonsillectomy, post-adenoidectomy, neurological and post-traumatic. Based on the findings on nasendoscopy, videofluoroscopy and clinical speech/voice analysis the following treatment options were recommended: 17 (40 per cent) for pharyngoplasty, five (11 per cent) for revision pharyngoplasty, 15 (35 per cent) for speech therapy, four for an obturator and one for tonsillectomy. Two previously undetected submucous clefts were diagnosed.

An algorithmic approach to aspergillus sinusitis.

The effective management of paranasal sinus aspergillosis requires early diagnosis, histological classification, surgery and where appropriate, chemotherapy. Fungal sinusitis may be easily missed unless a high index of suspicion is maintained and specific culture and histology requested. The disease is classified into invasive and noninvasive types, each being divided into two subgroups: invasive aspergillosis may be either fulminant or indolent and noninvasive disease localized or allergic. The literature is reviewed and an algorithmic approach to aspergillus sinusitis proposed. The importance of histologically differentiating invasive from noninvasive aspergillosis prior to selecting the appropriate treatment options is stressed. CT scan should precede definitive surgery, and be used in follow-up. Close and prolonged follow-up is essential.

Synovial sarcoma of the pharynx.

Synovial sarcoma is rarely seen in the head and neck region. A case of synovial sarcoma of the pharynx in a child is presented.

Relative bioavailability of two brands of ornidazole in twelve healthy volunteers.

AIM OF THE STUDY: To determine the bioequivalence of two marketed ornidazole formulations in healthy volunteers. METHODOLOGY: A single dose relative bioavailability of Ornidazole 1.5 g (3 x 500 mg tablets) of test product (Giro, Panacea Biotec Ltd.) and that of standard reference (Dazolic, Sun Pharmaceutical Industries), was investigated in healthy adult males. A total of 12 subjects wee enrolled in the study and investigations consisted of two treatment phases separated by a washout period of seven days. Both treatment phases were of 12 hours durations each. Blood samples were collected at 0, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10 and 12 hours post-administration of assigned drug product. Appropriate fasting restrictions were employed during both the treatment phases. Drug assay was done using HPTLC method. The statistical significance of difference in pharmacokinetic parameters between preparations was tested using ANOVA. RESULTS: The mean peak plasma concentration (Cmax) of 32.67 +/- 4.45 microg/ml was achieved at 1.54 +/- 0.81 hours following administration of test product as against mean Cmax of 31.55 +/- 5.04 microg/ml at 1.79 +/- 0.89 hours for reference standard. The area under time concentration curve (AUC(0-12)) hours was 261.67 +/- 77 microg/ml hours with reference standard and 265.41 +/- 30.82 microg/ml hours for test product. CONCLUSION: There was no statistically significant difference between the two formulations and the two products

Outcomes for patients referred urgently with suspected head and neck cancer.

INTRODUCTION: The 1998 National Health Service White Paper stated that anyone suspected of having a cancer would be seen by a specialist within two weeks. The 'trigger symptoms' prompting such referral have been nationally agreed by the National Institute for Health and Clinical Excellence. This study aimed to quantify the diagnostic yield of urgent referrals for suspected head and neck malignancy, and to identify reasons why patients ultimately diagnosed with malignancy may not have been referred via this pathway. MATERIALS AND METHODS: All patients referred to the trust with suspected head and neck malignancy in 2005 were included in the study. Data were obtained on date of referral, date of appointment, reason for referral and which National Institute for Health and Clinical Excellence guideline heading the referral fell under, clinical findings, and final diagnosis. Concurrently, all patients in the trust with a histological diagnosis of head and neck malignancy were identified using the computer records of the pathology department. RESULTS: One hundred and seventy-seven patients were referred with suspected head and neck malignancy over the one-year study period. Of these, 169 were seen within two weeks. The commonest causes of referral were hoarseness and neck lumps. Of these patients, 22 (12 per cent) were ultimately diagnosed with malignancy. During the one-year study period, 39 patients were diagnosed hospital-wide with head and neck malignancy, 17 of whom had not been referred via the urgent referral pathway. No unifying theme was identified to explain why these patients had not been referred via this pathway. CONCLUSION: In a group of patients with symptoms suggestive of head and neck malignancy, only 12 per cent were ultimately diagnosed with cancer. Of all the patients within the trust diagnosed with head and neck cancer, 44 per cent had come from outside the urgent referral pathway.

Role of otoacoustic emission in children with middle-ear effusion and grommets.

OBJECTIVE: To evaluate the role of otoacoustic emission in children with middle-ear effusion and grommets. MATERIALS AND METHODS: A prospective study was carried out on a total of 90 ears. All children listed for grommet insertion had a pre-operative and post-operative (three to six months after grommet insertion) pure tone audiometry, tympanometry and otoacoustic emission recorded. A comparison was made between pure tone audiometry and otoacoustic emission both pre-operatively and post-operatively. RESULTS: Pre-operatively, 63 ears had an abnormal pure tone audiometry of which 59 had absent otoacoustic emission. Therefore the sensitivity of otoacoustic emission in detecting a conductive loss was 59/63 = 94 per cent (95 per cent confidence interval 85 to 98 per cent). All 27 ears with normal hearing pre-operatively had normal otoacoustic emission. The specificity of otoacoustic emission was 27/27 = 100 per cent, (95 per cent confidence interval, 88 to 100 per cent). The positive predictive value was 59/59 = 100 per cent (95 per cent confidence interval, 94 to 100 per cent). After three to six months all post-operative patients with grommets had a normal pure tone audiometry and otoacoustic emission. So both pure tone audiometry and otoacoustic emission were strongly related both in patients with middle-ear effusion and in patients with grommets. CONCLUSION: As the demonstration of hearing in young and difficult-to-test children can be problematic and time-consuming, we suggest that otoacoustic emission can be used as an alternative to pure tone audiometry in patients with middle-ear effusion and grommets.

Epidermal inclusion cysts in the male breast.

The authors present the second and third reported cases of an epidermal inclusion cyst presenting as a breast mass in a male. In each of the two patients the mass appeared on mammography as a well-defined, noncalcified mass, and breast cancer was suspected. Biopsy was required to establish the correct diagnosis.

Mammography in men.

PURPOSE: To evaluate mammographic findings in men with abnormality of the breast. MATERIALS AND METHODS: The most common indications for mammography, performed in 263 men (mean age, 62 years; range, 19-93 years), were breast tenderness in 88 men (33%) and palpable mass in 79 men (30%). All chief symptoms and all mammographic findings were categorized. Biopsy results were correlated with mammographic findings in 24 patients. Follow-up ranged from 6 months to 8 years 4 months (mean, 3 years 4 months). RESULTS: The most common mammographic finding was gynecomastia in 213 men (81%). Gynecomastia was unilateral or asymmetric in 168 men (72%). Biopsy results in 14 patients with gynecomastia were negative for cancer; the 199 other patients did not have cancer at follow-up. Six patients had breast cancer: Four had primary breast cancer, and two had metastases to the breast from extramammary malignancies. No cancer had the mammographic appearance of gynecomastia. None of the 43 patients younger than age 50 years had cancer. CONCLUSION: A man with breast enlargement or a palpable mass who has the mammographic diagnosis of gynecomastia need not undergo biopsy unless there are other strong clinical indications [corrected]. A man younger than age 50 years with diffuse breast enlargement or a palpable, nonindurated, central, subareolar mass need not undergo mammography unless there are other strong clinical indications.

Formation and metabolism of histone mRNAs with mutant 3' ends formed by snRNA termination signals.

Histone mRNAs are the only non-polyadenylated mRNAs, ending in a conserved 26 nt sequence which can form a stem-loop. It has not been possible to make histone mRNAs with mutant stem-loops since most mutations in the stem-loop interfere with the 3' processing reaction. The snRNA genes transcribed by RNA polymerase II form their 3' ends by transcription termination directed by a signal which is located entirely 3' of the snRNA coding sequence. Chimeric genes which express RNAs ending in a histone 3' end or in mutant histone 3' ends formed by snRNA termination signals were constructed. The mRNAs from these genes were efficiently transported from the nucleus after injection of the genes into frog oocytes. This was true even for RNAs which end in mutant stem-loops suggesting that the snRNA termination signals promote transport of the transcripts.

Increasing the distance between the snRNA promoter and the 3' box decreases the efficiency of snRNA 3'-end formation.

Chimeric genes which contained the mouse U1b snRNA promoter, portions of the histone H2a or globin coding regions and the U1b 3'-end followed by a histone 3'-end were constructed. The distance between the U1 promoter and the U1 3' box was varied between 146 and 670 nt. The chimeric genes were introduced into CHO cells by stable transfection or into Xenopus oocytes by microinjection. The efficiency of utilization of the U1 3' box, as measured by the relative amounts of transcripts that ended at the U1 3' box and the histone 3'-end, was dependent on the distance between the promoter and 3'-end box. U1 3'-ends were formed with >90% efficiency on transcripts shorter than 200 nt, with 50-70% efficiency on transcripts of 280-400 nt and with only 10-20% efficiency on transcripts >500 nt. Essentially identical results were obtained after stable transfection of CHO cells or after injecting the genes into Xenopus oocytes. The distance between the U1 promoter and the U1 3' box must be <280 nt for efficient transcription termination at the U1 3' box, regardless of the sequence transcribed.

Targeted disruption of the CP2 gene, a member of the NTF family of transcription factors.

The NTF-like family of transcription factors have been implicated in developmental regulation in organisms as diverse as Drosophila and man. The two mammalian members of this family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related proteins sharing an overall amino acid identity of 72%. CP2, the best characterized of these factors, is a ubiquitously expressed 66-kDa protein that binds the regulatory regions of many diverse genes. Consequently, a role for CP2 has been proposed in globin gene expression, T-cell responses to mitogenic stimulation, and several other cellular processes. To elucidate the in vivo role of CP2, we have generated mice nullizygous for the CP2 allele. These animals were born in a normal Mendelian distribution and displayed no defects in growth, behavior, fertility, or development. Specifically, no perturbation of hematopoietic differentiation, globin gene expression, or immunological responses to T- and B-cell mitogenic stimulation was observed. RNA and protein analysis confirmed that the nullizygous mice expressed no full-length or truncated version of CP2. Electrophoretic mobility shift assays with nuclear extracts from multiple tissues demonstrated loss of CP2 DNA binding activity in the -/- lines. However, a slower migrating complex that was ablated with antiserum to NF2d9, the murine homologue of LBP-1a, was observed with these extracts. Furthermore, we demonstrate that recombinant LBP-1a can bind to known CP2 consensus sites and form protein complexes with previously defined heteromeric partners of CP2. These results suggest that LBP-1a/NF2d9 may compensate for loss of CP2 expression in vivo and that further analysis of the role of the NTF family of proteins requires the targeting of the NF2d9 gene.

The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the alpha- and beta-globin gene loci in an erythroid cell line.

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human beta-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous beta- or alpha-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

Regions of microsynteny in Magnaporthe grisea and Neurospora crassa.

A bacterial artificial chromosome (BAC) clone containing 110,467 bp of genomic DNA from Magnaporthe grisea was sequenced, annotated, and compared to the genomes of Neurospora crassa, Candida albicans, and Saccharomyces cerevisiae. Twenty-six open reading frames (ORFs), involved in multiple biochemical pathways, were identified in the BAC sequence. A region of 53 kb, containing 18 of the 26 ORFs, was found to be syntenic to a portion of the N. crassa genome. Subregions of complete colinearity as well as interrupted colinearity were present. No synteny was evident with either C. albicans or S. cerevisiae. The identification of syntenic regions containing highly conserved genes across two genera that have been evolutionarily separated for approximately 200 million years elicits many biological questions as to the function and identity of these genes.

Gene discovery and gene function assignment in filamentous fungi.

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.