A Pneumatic Device for Rapid Loading of DNA Sequencing Gels

This work describes the design and construction of a device that facilitates the loading of DNA samples onto polyacrylamide gels for detection in the Perkin Elmer/Applied Biosystems (PE/ABI) 373 and 377 DNA sequencing instruments. The device is mounted onto the existing gel cassettes and makes the process of loading high-density gels less cumbersome while the associated time and errors are reduced. The principle of operation includes the simultaneous transfer of the entire batch of samples, in which a spring-loaded air cylinder generates positive pressure and flexible silica capillaries transfer the samples. A retractable capillary array carrier allows the delivery ends of the capillaries to be held up clear of the gel during loader attachment on the gel plates, while enabling their insertion in the gel wells once the device is securely mounted. Gel-loading devices capable of simultaneously transferring 72 samples onto the PE/ABI 373 and 377 are currently being used in our production sequencing groups while a 96-sample transfer prototype undergoes testing.     

Maintenance and synthesis of proteins for an anucleate axon

The anucleate (distal) segment of a crayfish medial giant axon (MGA) remains intact for months in vivo after severing the axon from its cell body, a phenomenon referred to as long-term survival (LTS). We collected axoplasm from chronic anucleate MGAs by perfusing 2-cm lengths of axons with an intracellular saline. This axoperfusate was analyzed by SDS-PAGE and silver stained. Axoperfusate proteins from intact MGAs and from chronic anucleate MGAs exhibiting LTS for up to 6 months were the same. Furthermore, immunoreactive levels of actin and β-tubulin were similar in axoperfusates from intact and chronic anucleate MGAs. This maintenance of proteins in chronic anucleate MGAs must be due to a lack of protein degradation and/or to local protein synthesis by a source other than the cell body. To investigate local protein synthesis in vitro, we added [³⁵S]-methionine to the extracellular saline surrounding intact and chronic anucleate MGAs. After 4- to 6-h incubations, radiolabelled proteins were detected in axoperfusates analyzed by SDS-PAGE and fluorography. The similarity between radiolabelled proteins in axoperfusates and MGA glial sheaths indicated a glial origin for the radiolabelled axoperfusate proteins. Various observations and control experiments suggested that glial-axonal protein transfer occurred by a physiological process. Glial-axonal protein transfer may contribute to the maintenance of proteins during LTS of chronic anucleate MGAs.     

Epidural abscess following epidural steroid and local anaesthetic injection

Epidural abscess is a well-recognised but rare complication of epidural catheter placement. We have found only five previous reports of epidural abscess from noncatheter-related administration of steroids and/or local anaesthetic. We describe a further case which led to critical illness and emphasise the association between diabetes mellitus and epidural infection.     

Early developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure decreases chick embryo heart chronotropic response to isoproterenol but not to agents affecting signals downstream of the beta-adrenergic receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes cardiovascular toxicity in laboratory animals, including alteration in several processes in which beta-adrenergic receptor (beta-AR) signaling plays important roles. Thus, our laboratory investigated the effects of TCDD on beta-AR expression and signal transduction. Fertile chicken eggs were injected with vehicle (corn oil), 0.24 or 0.3 pmol TCDD/g egg on incubation day 0 (D0) or D5. On D10, heart function was assessed by ECG in ovo. Exposure to TCDD increased the incidence of arrhythmias and decreased the positive chronotropic responsiveness of the heart to isoproterenol. The reduced beta-AR responsiveness was, in part, independent of any overt morphological changes in the heart as chick embryos exposed to TCDD on D5 displayed an intermediate responsiveness to beta-AR agonist in the absence of the dilated cardiomyopathy observed in chick embryos exposed to TCDD on D0. TCDD did not decrease the chronotropic response of the heart to agents that stimulate signals downstream of the beta-AR. In fact, TCDD-exposed embryos were more sensitive than controls to forskolin, increasing heart rates (HR) 21.8 +/- 3.5 beats per min (bpm) above baseline versus control values at 6.3 +/- 2.7 bpm above baseline. TCDD exposure also augmented the negative chronotropic response of the heart to verapamil, decreasing HR -23.2 +/- 7.4 bpm relative to baseline versus control embryos at -12.7 +/- 5.9 bpm below baseline. Finally, the mean cardiac beta1-AR mRNA expression in D10 embryos was not significantly altered by exposure to TCDD on D0. These findings establish that a functional end point of the developing chick heart is sensitive to TCDD exposure and that the TCDD-induced reduction in beta-AR responsiveness may result from alterations in signal transduction upstream of adenylyl cyclase.     

Public–Private Collaboration in Health and Human Service Delivery: Evidence from Community Partnerships

The collaboration among public–private partnerships that applied to the Community Care Network (CCN) demonstration program of the Hospital Research and Educational Trust is examined. These partnerships link broad-based community coalitions with health and human service providers in efforts to improve community health and local service delivery. Although they willingly collaborated in identifying community health needs, coordinating services, and reporting to the community, partnership participants showed less alacrity in joining forces to reduce redundancy and increase efficiency. Such patterns suggest that organizations might best profit from working together on activities that maintain existing power relations and that have the potential to add prestige and attract new clients. Collaboration in these areas may be essential to building a foundation of trust that leads to future cooperation in more sensitive areas.     

HLA-DR expression in macaque neuroendothelial cells in vitro and during SIV encephalitis

HLA-DR expression in neuroendothelial cells (NEC) was studied during the course of SIV encephalitis in rhesus monkeys. HLA-DR determinants were detected on NEC in monkeys with SIV encephalitis, but not in control animals. In situ hybridization with an SIV probe indicated that HLA-DR expression was not a consequence of SIV replication within NEC. Cultured rhesus NEC stimulated with gamma interferon expressed HLA-DR to a higher degree than cultured brain fibroblasts or astrocytes. These data support the contention that NEC participate in retrovirus-induced inflammation and autoimmunity within the central nervous system.     

Genetic determinants of statin intolerance

Background  

Statin-related skeletal muscle disorders range from benign myalgias – such as non-specific muscle aches or joint pains without elevated serum creatinine kinase (CK) concentration – to true myositis with >10-fold elevation of serum CK, to rhabdomyolysis and myoglobinuria. The genetic basis of statin-related muscle disorders is largely unknown. Because mutations in the COQ2 gene are associated with severe inherited myopathy, we hypothesized that common, mild genetic variation in COQ2 would be associated with inter-individual variation in statin intolerance. We studied 133 subjects who developed myopathy on statin monotherapy and 158 matched controls who tolerated statins without incident or complaint.     

Right superficial femoral artery to superior vena cava graft using a polytetrafluroethylene graft: a new technique in a complicated dialysis access patient.

Sir, The expansion of haemodialysis includes older patients withcomorbidities, poor quality vessels, unsuitable for transplantationor peritoneal dialysis. Vascular access complications accountfor 20% of hospital admissions [1]. With exhausted sites inupper extremities, unusual sites for arteriovenous grafts formationare used [2]. The