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.     

Controlling bacterial contamination of an enteral formula through the use of a unique closed system: contamination, enteral formulas, closed system

Contamination of enteral diets may play an essential role in formula tolerance and safety for patients. Contaminated enteral formula commonly support microbiological growth. Commercially sterile liquid formulas received from the manufacturer are required by the Food & Drug Administration (FDA) to be shelf-stable and free from enteric pathogens. This study examined the use of large volume, closed system containers in a typical nursing home. Large volume (1500 mL) containers with unique pierceable caps and piercing spikes were studied to determine their ability to reduce the incidence of microbiological contamination due to their design and ability to decrease handling requirements. This study took place in a room of a typical nursing home. In this clinical setting, 211 containers and administration spike sets were evaluated following a 36-h hangtime. Contamination was virtually nondetectable. Nursing staff in a clinical facility can effectively utilize a large volume, prefilled, ready-to-use feeding system to achieve delivery of noncontaminated product for up to 36 h hangtime.     

Primary focal segmental glomerulosclerosis: clinical course and response to therapy

Primary focal segmental glomerulosclerosis (FSGS) is a lesion associated with a poor prognosis and results in end-stage renal disease after 5 to 10 years. Based on past experience, many nephrologists have considered primary FSGS a lesion that is steroid resistant and therefore are reluctant to offer steroids as treatment. Recent data, however, have demonstrated that patients with primary FSGS have a response to steroid therapy that is considerably better than had been described. Thus, it may be that nephrologists have been more "steroid reluctant" than the lesion is steroid resistant. To better understand this issue we review the clinical course and response to therapy in patients with primary FSGS.     

Disruption of epithelial cell-matrix interactions induces apoptosis

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Programmed cell death (apoptosis) is crucial for maintaining appropriate cell number and tissue organization. It was therefore of interest to determine whether cell-matrix interactions affect apoptosis. The present report demonstrates that apoptosis was induced by disruption of the interactions between normal epithelial cells and extracellular matrix. We have termed this phenomenon "anoikis." Overexpression of bcl-2 protected cells against anoikis. Cellular sensitivity to anoikis was apparently regulated: (a) anoikis did not occur in normal fibroblasts; (b) it was abrogated in epithelial cells by transformation with v-Ha-ras, v-src, or treatment with phorbol ester; (c) sensitivity to anoikis was conferred upon HT1080 cells or v-Ha-ras-transformed MDCK cells by reverse-transformation with adenovirus E1a; (d) anoikis in MDCK cells was alleviated by the motility factor, scatter factor. The results suggest that the circumvention of anoikis accompanies the acquisition of anchorage independence or cell motility.     

Transfer of loads between lumbar tissues during the flexion-relaxation phenomenon

STUDY DESIGN AND METHODS: This study used an anatomically detailed model of the lumbar tissues, driven from biologic signals of vertebral displacement and myoelectric signals, to estimate individual muscle and passive tissue force-time histories during the performance of the "flexion-relaxation" maneuver. Eight male university students performed three trials each of the "flexion-relaxation" maneuver with six pairs of surface myoelectric electrodes monitoring the right side of the trunk musculature, an electromagnetic device to record lumbar flexion, and videotape to record body segment displacement. OBJECTIVES: To examine the loads on individual tissues during the transfer of moment support responsibility from predominantly active muscle to predominantly passive tissue. SUMMARY OF BACKGROUND DATA: No previous studies, to the authors' knowledge, have examined individual tissue loading during the flexion-relaxation maneuver. RESULTS: Although most subjects were able to "relax" their lumbar extensors in full flexion, activity remained in the thoracic extensors and abdominals. Tissue load predictions suggested that while the lumbar extensor muscles were neurally "relaxed" (i.e., myoelectric silence), substantial elastic forces would assist the passive tissues in extensor moment support. On average, subjects sustained almost 3 kN in compressive load on the lumbar spine and about 755 N of anterior shear during full flexion with only 8 kg held in the hands. CONCLUSIONS: The "relaxation" of lumbar extensor muscles appeared to occur only in an electrical sense because they generated substantial force elastically through stretching. Loading of the interspinous and supraspinous ligaments, in particular, was high relative to their failure tolerance.     

The role of autologous blood transfusion in the the Federal Republic of Germany. Results of a 1993 questionnaire. 1. The reunited Germany

The objective of the study was to evaluate the role of autologous blood transfusion in current clinical practice in the Federal Republic of Germany after reunification. METHODS: Questionnaires were sent to the anaesthesia departments of 684 German hospitals in January 1993. The sample consisted of 400 randomly selected hospitals from the former West Germany ("old federal states") and 284 hospitals from the former German Democratic Republic ("new federal states"). Only hospitals with more than 25 surgical beds were included in the study. The questionnaire contained 36 questions related to (1) general information on the hospital, (2) preoperative autologous blood donation (PABD), (3) preoperative plasmapheresis, (4) isovolaemic haemodilution, (5) perioperative blood salvage, and (6) general management of blood transfusion. RESULTS: A total of 502 completed questionnaires (73%) were returned, 305 from hospitals in the "old federal states" and 197 from hospitals in the former German Democratic Republic. Nine per cent of the responding hospitals were running their own transfusion services, and 56% were located in the vicinity of a regional blood bank. The overall proportion of surgical procedures requiring perioperative blood transfusion ranged from 1% to 90% (median 10%). PABD was performed "not at all" in 18%, "rarely" in 20% "occasionally" in 27%, "frequently" in 17%, and "mostly" in 16% of the responding hospitals. The principal use of PABD was in orthopaedic surgery and cardiac surgery (83% and 70% of the departments in question, respectively). In more than 50% of the hospitals reporting, the PABD service was run by the anaesthesia department. Patients not meeting the established criteria for homologous blood donors were accepted for autologous blood donation "frequently" and "mostly" in 20% and 12% of the hospitals, respectively, but at most "occasionally" in 63% of the hospitals. Preoperative plasmapheresis was performed in 12% of the responding hospitals. Autologous fresh frozen plasma predominantly was used for volume replacement, and for prevention of coagulation disorders when major blood loss was anticipated. Isovolaemic haemodilution was performed "not at all" in 28%, "rarely" in 19%, "occasionally" in 28%, "frequently" in 16%, and "mostly" in 8% of the responding hospitals. The reasons most frequently invoked for not performing haemodilution were "too time consuming" and "too little blood-saving effect". Cell separators for perioperative blood salvage were available in 30% of the responding hospitals. Of the other hospitals not equipped with cell-washing devices, 11% performed perioperative blood salvage of unprocessed blood by means of simple collection devices. Some 80% of those hospitals using intraoperative autotransfusion devices also performed blood salvage postoperatively. The principal use of perioperative blood salvage was in cardiac surgery, orthopaedics, and vascular surgery (90%, 54%, and 54% of departments, respectively). Some 48% of the responding anaesthetists "mostly" considered haemoglobin levels of 8-10 g/dl acceptable in patients without cardiopulmonary disease, but only 18% did so in patients with cardiopulmonary disease. CONCLUSIONS: Although available in the majority of hospitals surveyed, the simple techniques of both PABD and isovolaemic haemodilution are unduly neglected in routine clinical practice. The consistent use of both of these techniques, and the careful weighing up of the indication for every single blood transfusion, would not only effectively reduce homologous blood transfusions, but also enable even small hospitals to run successful autologous transfusion programmes without expensive cell-washing and plasmapheresis devices.     

Strategic questions for consumer-based health communications

Using the consumer-oriented approach of social and commercial marketers, this article presents a process for crafting messages designed to improve people's health behaviors. The process, termed consumer-based health communications (CHC), transforms scientific recommendations into message strategies that are relevant to the consumer. The core of CHC is consumer research conducted to understand the consumer's reality, and thereby allowing six strategic questions to be answered. The immediate result of the CHC process is a strategy statement--a few pages that lay out who the target consumer is, what action should be taken, what to promise and how to make the promise credible, how and when to reach him or her, and what image to convey. The strategy statement then guides the execution of all communication efforts, be they public relations, mass media, direct marketing, media advocacy, or interpersonal influence. It identifies the most important "levers" for contact with the consumer. Everyone from creative specialists through management and program personnel can use the strategy statement as a touchstone to guide and judge the effectiveness of their efforts. The article provides a step by step illustration of the CHC process using the 5 A Day campaign as an example.