Expansion and length-dependent fragility of CTG repeats in yeast

Expansion of DNA trinucleotide repeats (TNRs) is the causative mutation in a growing number of human genetic diseases. Large expansions of a CTG tract were obtained and shown by genetic and physical assays to be length-dependent sites of chromosome breakage in Saccharomyces cerevisiae. Deletion of RAD27, which encodes a nuclease involved in Okazaki fragment processing, caused length-dependent destabilization of CTG tracts and a substantial increase in expansion frequency. The genetic assay described here can be used to evaluate other factors that induce TNR expansion or chromosome fragility in humans.     

Protein phosphatases and the regulation of MAP kinase activity

A family with hereditary factor X deficiency is presented. One member, a 25-year-old man, showed a mild bleeding tendency. His factor X activity (extrinsic: 56%; intrinsic: 55%; Russell's viper venom: 57%) and his level of circulating factor X antigen (55% of normal) were markedly reduced. Analysis of his factor X gene revealed a single point mutation within exon II resulting in the substitution of +25 Gla (GAA) by Lys (AAA). The mutation was determined by gene analysis to be heterozygous in this patient, his mother and one of his brothers. Clotting assays of factor X purified from the plasma of the index patient revealed an activity of 89% of normal upon activation with Russell's viper venom, 77% of normal in the intrinsic and 81% of normal in the extrinsic coagulation pathway. The mutation responsible for the substitution of Lys for Gla+25 was introduced into an expression plasmid containing a wild type factor X cDNA and expressed in a mammalian cell line. Factor X antigen levels in the cell lysates and in the supernatant were identical in the mutant and wild type constructs. The specific activity of the factor X expressed from the mutant construct was 3% compared with the wild type construct. These data demonstrate that the substitution of Lys for Gla+25 results not only in a reduced level of factor X in the affected family members, but also in a substantial loss of specific factor X activity.     

Learning curve for oesophageal cancer surgery

BACKGROUND: Surgical training and experience are frequently claimed to influence early and late outcome measures. The aim of this study was to examine any improvement in an individual surgeon's performance in one operation over a period of 7 years from initial appointment to date. METHODS: Patients undergoing Ivor Lewis subtotal oesophagectomy performed by a single surgeon between April 1990 and December 1996 were identified from a prospectively compiled oesophageal cancer database. Operating time (abdominal, thoracic and 'one-lung time'), blood loss, transfusion requirements (intraoperative and total), extent of lymphadenectomy (number of lymph nodes sampled), intensive treatment unit (ITU) stay, hospital stay, postoperative morbidity and mortality, pathological stage, grade and survival were recorded. RESULTS: The records of 150 patients were identified for analysis. The cohort was split into five groups, each of 30 patients operated on consecutively. Each of the groups was comparable for age, sex, smoking history, preoperative haemoglobin and creatinine levels, weight loss, American Society of Anesthesiologists' grade, and histological stage and grade of disease. Analysis of the variables pertaining to operation revealed a significant improvement with time including reduced single-lung operating time (P=0.01), reduced blood loss (P=0.03), reduced transfusion requirement (P < 0.0001), reduced ITU stay (P< 0.0001), reduced inpatient stay (P< 0.0001) and an increased yield of lymph nodes (P < 0.0001). CONCLUSION: This study showed a continuing improvement in a surgeon's performance over a 7-year period. With the current trend to shorter training periods there is a case for continuing supervision of the 'fully trained' surgeon within highly specialist units.     

Current options in prosthetic vascular graft infection

Debate continues over which procedure is the best treatment for prosthetic graft infections. We retrospectively reviewed the medical records at our institution for all vascular graft infections that occurred from 1985 to 1995 to evaluate their occurrence, treatment, and outcome. Twenty-four patients had prosthetic graft infections. The average patient age was 62 years, and 67 per cent of the patients studied were men. The initial operation was for treatment of occlusive disease in 92 per cent of the patients, and aortofemoral bypasses were the most common procedures performed (15 of 24 patients, 63%). The average interval from graft implantation to presentation of infection was 29 months. In lower-extremity bypasses, the site of infection was most commonly in the groin (87%). Gram-positive organisms, including coagulase-negative Staphylococcus (32%) and Staphylococcus aureus (28%), were the most frequently isolated bacteria. Thirty procedures were performed for management of the graft infections. Extra-anatomic bypass was associated with no recurrent graft infections. Graft preservation was successful in two cases of early S. aureus infection (less than 1 year after original procedure), and in situ graft replacement was successful in all four cases of late-appearing coagulase-negative Staphylococcus infection (more than 1 year after original procedure). Both treatments failed in all five cases of Gram-negative infection (P = 0.008 by Fisher's exact test). The overall mortality and amputation rates were 17 per cent and 21 per cent, respectively, without significant differences between the treatment modalities. Extra-anatomic bypass remains the best treatment for prosthetic graft infection. In situ replacement and graft preservation treatments should be selective and based on presentation of the infection and the type of pathogenic organism.     

The detection of oligonucleotide N3'-->P5' phosphoramidate/RNA duplexes with capillary gel electrophoresis

Oligonucleotide N3'-->P5' phosphoramidates (3'-phosphoramidates) are DNA analogs that are presently under investigation as potential therapeutic agents. These compounds may also hold promise as a diagnostic tool. Here, we describe a rapid method for the analysis of single-stranded RNA fragments utilizing 3'-phosphoramidate oligonucleotides as probes in conjunction with capillary gel electrophoresis (CGE). 3'-Phosphoramidate 9-mers were mixed with complimentary RNA, and CGE was used to monitor duplex formation. Complimentary strands of RNA and 3'-phosphoramidate formed duplexes that gave unique relative mobilities based on an internal standard. The ability of CGE to discriminate between perfect duplexes and duplexes that contain a base mismatch was also investigated. However, the primary focus of this work was to determine CGE's ability to detect the presence of the 3'-phosphoramidates/RNA duplex under routine electrophoretic running conditions. Polyacrylamide gel electrophoresis analysis was utilized to verify duplex formation.     

Diverse Fab specific for acetylcholine receptor epitopes from a myasthenia gravis thymus combinatorial library

The muscle weakness in myasthenia gravis (MG) is caused by heterogeneous high-affinity IgG autoantibodies to the nicotinic acetylcholine receptor (AChR), a complex ion channel glycoprotein. These antibodies are clearly responsible for reducing AChR numbers at the neuromuscular junction in myasthenia; however, the origins, diversity, specificity and pathogenicity of individual antibodies have not yet been established. We have cloned and characterized four different AChR-specific Fab from an MG patient's thymus by screening an IgG1/kappa gene combinatorial lambda phage library with soluble human AChR labeled with [125I] alpha-bungarotoxin. Unlike most previously cloned human antibodies, all four Fab immunoprecipitated soluble human muscle AChR. Two Fab strongly inhibited binding of mAb to the main immunogenic region on the alpha subunits and one Fab bound to an epitope on the fetal-specific gamma subunit. In sensitivity and fine specificity, these Fab resembled the anti-AChR antibodies found in many MG patients, including the donor. The closest germline counterparts for their heavy chains were in VH families 1, 3 and 4; however, there were many differences consistent with an antigen-driven response of diverse B cell clones. The combinatorial approach holds promise for further analysis of human autoantibodies.     

Urine osmolality and conductivity as indices of hydration status in athletes in the heat

PURPOSE: The purpose of this study was to determine a quick and easy method for assessment of day-to-day hydration status in athletes in the heat. METHODS: Measurement of the osmolality of the first urine sample of the day collected after wakening but before breakfast established a standardized collection procedure to allow day-to-day comparisons of individuals. RESULTS: Laboratory measurements established that a difference in osmolality is found when individuals are dehydrated by a moderate extent in comparison with an euhydrated situation: the osmolality of the first morning urine sample of control subjects (N = 11) averaged over 5 d was 675 (+/- 232) mosmol.kg-1 (mean +/- SD). For subjects who were hypohydrated by exercise followed by fluid restriction, morning urine osmolality was 924 (+/- 99) mosmol.kg-1 (P < 0.001, N = 11, averaged over 7 d). Field measurements from 29 athletes undertaking warm weather training indicated that the athletes could, with appropriate feedback, maintain a satisfactory hydration status. Athletes in weight category sports tended to record a higher morning urine osmolality, reflecting their attempts to dehydrate: recorded values were 627 (+/- 186) mosmol.kg-1 (nonweight category sports, N = 8), 775 (+/- 263) mosmol.kg-1 (boxers, N = 15) and 777 (+/- 254) mosmol.kg-1 (wrestlers, N = 6). Results obtained with a hand-held portable conductivity were compared with those from measured osmolality. CONCLUSIONS: The findings suggest that such an instrument could provide athletes with reliable information as to their hydration status from measurement of the first morning urine of the day and therefore provide a quick and easy method for achieving an approximation of hydration status from day-to-day.