A view of the future: Dr. Mark Gilbert of Immunex talks about immune based therapy. Interview by Robert Nielsen.
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AIDS Vaccines↗
Biomedical subjects
M Gilbert
Publications and source records attributed to M Gilbert.
Hormonal counterregulation failure in rats is related to previous hyperglycaemia-hyperinsulinaemia.
Hyperglycaemia and hyperinsulinaemia were induced in rats by a continuous 48-h infusion with glucose. Discontinuation of glucose infusion resulted in marked, persistent hypoglycaemia. To further delineate the mechanism underlying this condition, we measured counterregulatory hormone levels, in vivo glucose kinetics (glucose production = rate of appearance = Ra; glucose utilization = rate of disappearance = Rd), and in vitro gluconeogenesis during the 48-h postinfusion period. Prior to cessation of glucose infusion, Rd was increased 6-fold when compared to control rats, whereas Ra was totally abolished. During the first hour after the end of glucose infusion, Ra increased and Rd decreased (but was still higher than Ra), inducing hypoglycaemia which stabilized after 1 h at ¿¿126¿¿3.5 mmol/l when both Ra and Rd became equal. Despite hypoglycaemia, plasma glucagon and catecholamine levels did not increase during the 3-to 36-h time interval. The increase in Ra during the first hour post-infusion was not related to changes in counterregulatory hormone response. The increase in glucose production was accounted for by glycogenolysis, as shown by total depletion in liver glycogen within 6 h and thereafter by gluconeogenesis. In vitro experiments using isolated hepatocytes suggested that gluconeogenesis was supported during the first 24 h by substrates entering the pathway beyond the step catalysed by the PEPCK enzyme. Thereafter, lactate became the major substrate, and this condition was associated with a progressive rise in glucagon concentration. It is concluded that 48 h of hyperglycaemia/hyperinsulinaemia resulted in a failure of counterregulatory hormonal response to hypoglycaemia. Yet, despite this lack of counterregulatory response, hepatic gluconeogenesis was stimulated in response to hypoglycaemia.
Animals↗
Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1 expression in bovine aortic endothelial cells.
To enlighten the mechanism of inhibition of prostacyclin (PGI2) production by n-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, cultured endothelial cells were incubated with albumin bound-EPA or -DHA for 22 h. Under these conditions, PGI2 formation in response to bradykinin, calcium ionophore or exogenous arachidonic acid was equally inhibited by 50%, suggesting that the inhibition might occur downstream the phospholipase step, likely at the level of PGH synthase and/or PGI2 synthase activities. Western blot analysis indicated that the mass of the constitutive isoform of PGH synthase (PGH synthase 1), but not PGI2 synthase, was significantly reduced in n-3 fatty acid-enriched cells. In subsequent experiments, PGH synthase 1 mRNA level, measured by northern blotting, was also decreased in n-3 supplemented cells. This reduction was not due to mRNA destabilization. None of these parameters were altered by similar enrichment with oleic acid (OA). These results suggest that EPA and DHA may affect PGH synthase 1 expression, presumably at the transcriptional level.
Animals↗
Characterization of a recombinant Neisseria meningitidis alpha-2,3-sialyltransferase and its acceptor specificity.
The structure and specificity of the recombinant alpha-2,3-sialyltransferase from Neisseria meninigitidis are reported. This enzyme showed an unusual acceptor specificity in that it could use alpha-terminal and beta-terminal Gal residues as acceptors. In addition (beta1-->4)-linked and (beta1-->3)-linked terminal Gal served as acceptors. These properties distinguish the bacterial enzyme from the more widely investigated mammalian equivalents. The protein was expressed as a membrane-associated protein in Escherichia coli at a level of 750 U/l (approximately 250 mg/l). The protein could be extracted with buffers containing 0.2% Triton X-100 and purified to homogeneity using immobilized-metal-affinity chromatography. Electrospray-ionization mass spectrometry of peptides obtained by cleavage with cyanogen bromide and trypsin confirmed over 95% of the deduced amino acid sequence. When used for enzymatic synthesis in coupled reactions with recombinant CMP-Neu5Ac synthetase, the alpha-2,3-sialyltransferase could sialylate fluorescent derivatives of N-acetyllactosamine with N-acetylneuraminic acid, N-propionylneuraminic acid and N-glycoloylneuraminic acid.
Amino Acid Sequence↗
Influence of Bcl-2 overexpression on Na+/K(+)-ATPase pump activity: correlation with radiation-induced programmed cell death.
Bcl-2 overexpression in transfected PW cells is associated with inhibition of radiation-induced programmed cell death (PCD). We have previously reported that there is a relationship between inhibition of radiation-induced PCD and membrane hyperpolarization in these cells. In this article, we report that Na+/ K(+)-ATPase pump activity, as measured by the uptake of Rubidium-86 (86Rb+), is significantly higher in Bcl-2 overexpressing PW cells than in control PW cells, and that pump activity following irradiation with doses > or = 500 cGy was reduced to a lesser extent in the Bcl-2 transfectants than in the control cells. When PW-Bcl-2 cells were incubated with a dose of ouabain (1 microM) that decreased pump activity significantly, but did not induce PCD, the previously reported protection from radiation-induced PCD associated with overexpression of Bcl-2 no longer existed. In order to demonstrate that reactive oxygen species (ROS) affected Na+/ K(+)-ATPase pump activity, cells were incubated with N-acetyl cysteine (NAC) prior to irradiation, or treated with the ROS generating drug buthionine sulphoxamine (BSO). 86Rb+ uptake was significantly higher in irradiated cells incubated with NAC compared to cells irradiated in the absence of NAC, while BSO resulted in lower levels of 86Rb+ uptake, suggesting that the effects of radiation on the Na+/K(+)-ATPase pump were due to ROS. Furthermore, the resting cell membrane potential of cells exposed to NAC were slightly hyperpolarized compared to control PW cells, whereas cells exposed to BSO were depolarized in comparison to control PW cells. In summary, this data suggests that Bcl-2 affects Na+/K(+)-ATPase pump activity, which is associated with the resting membrane potential and the level of susceptibility to radiation-induced PCD.
Apoptosis↗
Isolated tears of the triangular fibrocartilage: management by early arthroscopic repair.
To evaluate the efficacy of arthroscopic repair of the triangular fibrocartilage complex (TFCC) tears treated within 4 months after injury, functional outcome after repair was determined following arthroscopic repair in 24 patients. The patients' average age was 31 years (range, 22-38 years); the average follow-up period was 34 months (range, 26-48 months). All patients had wrist pain limiting their participation in work prior to surgery. Patients with central attrition tears identified by arthroscopy were excluded from the study. Twenty-three patients had a preoperative arthrogram. Twelve of the patients with positive arthrogram findings had an avulsion of the TFCC from the sigmoid notch (Palmer type 1D tears). Of the eleven patients with negative arthrograms, 10 had ulnar tears in capsular attachments of the TFCC. The ulna variance averaged 0.2 mm +/- 0.6 mm. Separate arthroscopic techniques were developed for reattaching the TFCC to the radius (nine patients) versus to the peripheral capsule on the volar or ulnar side of the wrist (eight patients). Postoperatively, there was a significant relief of pain (p < .01). Postoperative range of motion averaged 89% +/- 9% SD of the contralateral side, and grip strength averaged 85% +/- 20% SD of the contralateral side. Thirteen of the 19 patients returning to work did so in their original jobs.
Adult↗
Failure of cast immobilization for thumb ulnar collateral ligament avulsion fractures.
To determine if small avulsion fractures of the thumb ulnar collateral ligament (UCL) with minimal (< or = 2.0 mm) displacement can successfully be treated by cast immobilization, the authors reviewed 9 patients with minimally displaced fractures initially treated by casting. Despite immobilization within an average of 2 days of the initial injury (range, 0-6 days), a minimum of 6 weeks of immobilization in a cast, and adequate rehabilitation, all 9 patients had persistent thumb pain, especially with activities requiring strong pinch. After undergoing open reduction and internal fixation, the patients had relief of thumb pain and pinch strength improved from 36% of the contralateral side to 89% (p < .01). Grip strength increased from 77% to 93% (p < .05), but the ranges of motion of the thumb metacarpophalangeal and interphalangeal joints were not significantly altered. Minimally displaced UCL avulsion fractures frequently have significant rotation that prevents successful fracture healing even with prompt cast immobilization.
Adolescent↗
Ulnar shortening combined with arthroscopic repairs in the delayed management of triangular fibrocartilage complex tears.
The functional outcome after surgery was determined in 21 patients an average of 29 months (range, 24-52 months) after surgery to evaluate the efficacy of arthroscopic repair of triangular fibrocartilage complex (TFCC) tears and ulnar shortening. All of the patients had reparable lesions of the TFCC treated after a delay of more than 6 months from the time of injury. The patients' average age was 32 years and all patients had wrist pain limiting them from work and/or sports prior to surgery. After surgery, there was a significant relief of pain (p < .01). Grip strength and range of motion averaged 83% +/- 18% and 81% +/- 16%, respectively, of that of the uninjured side. At follow-up evaluation, 14 patients with repairs underwent follow-up studies; the TFCC was noted to be intact in 12 patients.
Adult↗
Animal assisted therapy and the individual with spinal cord injury.
Spinal cord injury (SCI) is a devastating event that results in significant adjustments during the acute and rehabilitation phase. During this period, it is imperative to maintain the patient's self-esteem, reduce stress levels, encourage the expression of feelings, and provide sensory stimulation. Animal Assisted Therapy (AAT) involves the use of animals as a complement to more traditional forms of therapy. The program is based on the knowledge that animals have a positive influence on people who are ill in the healthcare setting. The Animals Heal Hearts Program (TM) has two components, pet visitation and pet therapy. Pet visitation consists of allowing a patient to have his/her own personal dog for a visit, provided there are no medical contraindications. Pet therapy is a structured program using a dog that has completed behavioral and health screening. Dogs are used in the hospital to reduce patients' stress, increase their self-esteem, and help them express feelings. The dogs provide sensory stimulation as patients view and handle the animals and learn about animals and pets. A carefully planned and evaluated program ensures that it is safe and effective.
Adaptation, Psychological↗
TPA induces apoptosis in MPC-11 mouse plasmacytoma cells grown in serum-free medium.
Apoptotic-like events could be rapidly induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cells of the mouse plasmacytoma cell line MPC-11 grown in serum-free medium. Indicators for apoptosis were morphological changes visualized by light and electron microscopy, such as chromatin condensation and the formation of cellular buds and fragments, as well as biochemical indices like the appearance of the so-called 'DNA ladder'. Additionally, in these cells which are usually devoid of significant amounts of cytoplasmic intermediate filament (cIF) proteins, synthesis and accumulation of the cIF protein vimentin was rapidly induced by TPA treatment and almost all cells became vimentin-positive. Later on, substantial amounts of vimentin and lamin B degradation products appeared, and an increasing fraction of cells displayed low or even undetectable quantities of intact vimentin. This subpopulation was characterized via microscopy to be in the late stages of apoptosis. We suggest that in MPC-11 cells undergoing apoptosis in response to TPA treatment vimentin as well as lamin B are degraded, leading to a rearrangement and eventual loss of their respective filament networks.
Animals↗
Cloning of the lipooligosaccharide alpha-2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae.
The genes encoding the alpha-2,3-sialyltransferases involved in lipooligosaccharide biosynthesis from Neisseria meningitidis and Neisseria gonorrhoeae have been cloned and expressed in Escherichia coli. A high sensitivity enzyme assay using a synthetic fluorescent glycosyltransferase acceptor and capillary electrophoresis was used to screen a genomic library of N. meningitidis MC58 L3 in a "divide and conquer" strategy. The gene, denoted lst, was found on a 2. 0-kilobase fragment of DNA, and its sequence was determined and then used to design probes to amplify and subsequently clone the corresponding lst genes from N. meningitidis 406Y L3, N. meningitidis M982B L7, and N. gonorrhoeae F62. Functional sialyltransferase was produced from the genes derived from both L3 N. meningitidis strains and the N. gonorrhoeae F62. However, the N. meningitidis M982B L7 gene contained a frameshift mutation that renders it inactive. The expression of the lst gene was easily detected using the enzyme assay, and the protein expression could be detected when an immunodetection tag was added to the COOH-terminal end of the protein. Using the synthetic acceptor N-acetyllactosamine-aminophenyl-(6-(5-(fluorescein-carboxamido)-hexan oic acid amide), the alpha-2,3 specificity of the enzyme was confirmed by NMR examination of the reaction product. The enzyme could also use synthetic acceptors with lactose or galactose as the saccharide portion. This study is the first example of the cloning, expression, and examination of alpha-2,3-sialyltransferase activity from a bacterial source.
Amino Acid Sequence↗
Cloning and sequencing of the secY homolog from Streptomyces lividans 1326.
Two conserved regions of SecY proteins from six Gram+ bacteria were exploited in a PCR-based strategy for isolating a secY homolog from Streptomyces lividans (Sl). The nucleotide sequence of part of a 3.8-kb fragment showed that the secY homolog is flanked, at the 5' end, by the gene encoding ribosomal protein L15 and, at the 3' end, by an adenylate kinase-encoding gene. The deduced gene product of secY would have 437 amino acids (aa) and an M(r) of 47,200. Sl SecY shows 89.5, 56.1, 42 and 40% identity to its homologs from Streptomyces scabies, Brevibacterium flavum, Bacillus subtilis and Escherichia coli, respectively. Promoterprobe analyses indicated that the secY gene probably contains its own promoter.
Amino Acid Sequence↗
Cloning of a secA homolog from Streptomyces lividans 1326 and overexpression in both S. lividans and Escherichia coli.
We cloned a gene encoding a SecA homolog from Streptomyces lividans 1326, a Gram-positive bacterium known to produce large amounts of extracellular proteins. A protein sequence alignment with the other bacterial SecA homologs revealed that S. lividans SecA shares from 39.5 to 44% identity with them, while it shares 34.2 to 37.2% identity with SecA homologs from plastids of algae and plants. We overexpressed the secA gene in S. lividans 1326 and Escherichia coli MM52 and in both cases we observed the production of a protein with an apparent molecular mass of 117.4 kDa. Although S. lividans SecA is similar to E. coli SecA, it does not complement a thermosensitive mutation in the E. coli secA gene. However, a hybrid polypeptide consisting of the N-terminal portion (first 242 amino acids) of the S. lividans SecA and the C-terminal portion (657 a.a.) of the wild-type E. coli SecA was able to complement this mutant.
Adenosine Triphosphatases↗
Managing the increased workload in anticoagulant clinics. Computerised decision support is valuable.
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Ambulatory Care Facilities↗
Chronic free fatty acid infusion in rats results in insulin resistance but no alteration in insulin-responsive glucose transporter levels in skeletal muscle.
To investigate the mechanism by which free fatty acids (FFA) affect glucose uptake, we studied the effect of chronic elevation (24 h) of plasma FFA in rats on whole body glucose disposal and glucose utilization index (GUI) in the basal state and under a euglycemic hyperinsulinemic clamp in relation to the amount of insulin-responsive glucose transporter (IRGT, i.e., GLUTU) protein in different muscles (oxidative and glycolytic) and adipose tissue. Infusion of intralipid in the basal state led to a approximately 40% increase in whole body glucose uptake and a approximately 250% increase in GUI in adipose tissue as compared to control rats. There was no change in the amount of IRGT protein in any of the muscle types whereas in fat depots it was either unchanged or decreased. Under moderate of supraphysiological hyperinsulinemia, increment of whole body glucose disposal was significantly lower in intralipid perfused rats when compared to controls (approximately 110 microU/mL: 0.7 +/- 0.1 vs. 1.3 +/- 0.1 mg/min, P < 0.02; approximately 1000 microU/mL: 3.0 +/- 0.2 vs. 3.9 +/- 0.4 mg/min, P < 0.02). Under moderate hyperinsulinemia stimulation, GUI was significantly reduced in different muscles and adipose tissue as compared to controls. We conclude that peripheral insulin resistance which occurs after elevation of plasma FFA levels does not seem to be explained by changes in the amount of IRGT protein in either oxidative or glycolytic skeletal muscle. Thus fatty acid infusion appears to be associated with a defect in IRGT translocation to the plasma membrane, fusion with the membrane, or intrinsic activity.
Adipose Tissue↗
Arthroscopic repair of the triangular fibrocartilage complex.
To evaluate the efficacy of arthroscopic repair of the triangular fibrocartilage complex (TFCC), functional outcome was determined after arthroscopic repair of 22 wrists in 21 patients. Average follow-up was 36 months (range, 26 to 48 months) and the patients' average age was 30 years (range, 22 to 38 years). All patients had wrist pain limiting them from work or any sports. Twelve of the 14 patients with positive arthrograms had an avulsion of the TFCC from the sigmoid notch (Palmer type 1D tears). Of the 6 patients with negative arthrograms, 5 had peripheral tears (Palmer type 1B). Five patients had associated lunotriquetral ligament repairs and 7 had ulna shortening osteotomies. There was a significant relief of pain and increase in work and sports activities (P < .01). Postoperative range of motion averaged 86% +/- 9% of the contralateral side and grip strength averaged 82% +/- 20% of the contralateral side. There was a significant correlation between the delay from injury to surgical repair and the final total range of motion and grip strength. Follow-up studies in 15 patients found that the repairs were intact in 12. Arthroscopic repair results in significant relief of pain and an increase in the ability to perform at work or sports.
Adult↗
Maintaining the balancing act: restructuring at the unit level.
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Hospital Restructuring↗
Pallidotomy: a surgical intervention for control of Parkinson's disease.
Parkinson's disease, a chronic incurable disorder, has a significant impact on quality of life. Parkinson's disease results from chronic degeneration of cells in the basal ganglia that produce dopamine. It typically affects elderly individuals producing muscle rigidity and akinesia. Traditionally, Parkinson's disease has been controlled by medications. However, for some patients, medications may no longer be effective, or may produce drug-related complications. For this group of patients, pallidotomy is gaining support as an intervention to control the debilitating symptoms of Parkinson's disease, thus enabling a person to perform everyday tasks. Although the procedure was developed in the 1950s, contemporary technology has allowed a resurgence of the intervention. With the focus on relief of two of the major symptoms of Parkinson's disease, the pallidotomy procedure is gaining support as a surgical intervention for this disease. Therefore, it is important for the health care team to have an understanding of the disease process, surgical intervention and postoperative care to enhance quality outcomes for this patient population.
Globus Pallidus↗