Identification of an insulin-responsive, slow endocytic recycling mechanism in Chinese hamster ovary cells

In adipocytes, the insulin-regulated aminopeptidase (IRAP) is trafficked through the same insulin-regulated recycling pathway as the GLUT4 glucose transporter. We find that a chimera, containing the cytoplasmic domain of IRAP fused to transmembrane and extracellular domains of the transferrin receptor, is slowly recycled and rapidly internalized in Chinese hamster ovary cells. Morphological studies indicate that the chimera is slowly trafficked through the general endosomal recycling compartment rather than being sorted to a specialized recycling pathway. A chimera in which a di-leucine sequence within the cytoplasmic domain of IRAP has been mutated to alanines is rapidly internalized and rapidly recycled, indicating that this di-leucine is required for the slow recycling but not for the rapid internalization. Insulin stimulates a 2-3-fold increase in the recycling of the chimera and only a 1.2-fold increase in the recycling of the transferrin receptor. The effect of insulin on the recycling of the chimera is blocked by wortmannin, a phosphatidylinositol 3'-kinase inhibitor. GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) increases the recycling of the chimera by 50% but has no effect on the recycling of the transferrin receptor. In these studies, we have identified in Chinese hamster ovary cells a novel, slow endocytic recycling mechanism that is regulated by insulin.     

Campylobacter enteritis in Johannesburg

In order to establish a reasonable protocol for a diagnostic laboratory we conducted a survey during which we confined the routine culture of stool samples for Campylobacter fetus to two groups--all infants under 2 years of age, and older children and adults with obviously diarrhoeic stools. Camp. fetus was isolated from 100 of 2323 stool specimens (4,3%). This is within the 3 - 8% isolation rates previously reported from surveys in which all specimens were cultured. Camp. fetus isolates represented 16,9% of all bacterial pathogens isolated, and Black infants showed a significantly greater isolation rate than White infants. We feel that culture for Camp. fetus is an essential part of any routine bacteriological investigation of diarrhoea. A partially selective culture policy for Camp. fetus will result in a recovery rate at least equal to that of Salmonella and enteropathogenic Escherichia coli.     

Nitrosation of dialkylamines in the presence of bile acid conjugates

The rate of nitrosation of dihexylamine is enhanced when the reaction is carried ut in the presence of micelles containing bile acid conjugates or bile acid conjugates and lecithin. The nitrosation of smaller dialkylamines is not affected. The nitrosation of dihexylamine under these conditions is inhibited by alpha-tocopherol at pH 3 or pH 5. At pH 3 the reaction is inhibited by ascorbic acid, while at pH 5 it is inhibited by equimolar concentrations of ascorbic acid, but accelerated by higher concentration of ascorbic acid. The effect of ascorbic acid at pH 5 depends on the presence of oxygen and sulfamate.     

TIMP-1 contact sites and perturbations of stromelysin 1 mapped by NMR and a paramagnetic surface probe

Surfaces of the 173 residue catalytic domain of human matrix metalloproteinase 3 (MMP-3(DeltaC)) affected by binding of the N-terminal, 126 residue inhibitory domain of human TIMP-1 (N-TIMP-1) have been investigated using an amide-directed, NMR-based approach. The interface was mapped by a novel method that compares amide proton line broadening by paramagnetic Gd-EDTA in the presence and absence of the binding partner. The results are consistent with the X-ray model of the complex of MMP-3(DeltaC) with TIMP-1 (Gomis-Rüth et al. (1997) Nature 389, 77-81). Residues Tyr155, Asn162, Val163, Leu164, His166, Ala167, Ala169, and Phe210 of MMP-3(DeltaC) are protected from broadening by the Gd-EDTA probe by binding to N-TIMP-1. N-TIMP-1-induced exposure of backbone amides of Asp238, Asn240, Gly241, and Ser244 of helix C of MMP-3(DeltaC) to Gd-EDTA confirms that the displacement of the N-terminus of MMP-3(DeltaC) occurs not only in the crystal but also in solution. These results validate comparative paramagnetic surface probing as a means of mapping protein-protein interfaces. Novel N-TIMP-1-dependent changes in hydrogen bonding near the active site of MMP-3(DeltaC) are reported. N-TIMP-1 binding causes the amide of Tyr223 of MMP-3(DeltaC) bound by N-TIMP-1 to exchange with water rapidly, implying a lack of the hydrogen bond observed in the crystal structure. The backbone amide proton of Asn162 becomes protected from rapid exchange upon forming a complex with N-TIMP-1 and could form a hydrogen bond to N-TIMP-1. N-TIMP-1 binding dramatically increases the rate of amide hydrogen exchange of Asp177 of the fifth beta strand of MMP-3(DeltaC), disrupting its otherwise stable hydrogen bond.     

Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.     

Spatial gradients of cytosolic calcium concentration in neurones during paradoxical activation by calcium

PURPOSE: To assess long-term survival following cladribine salvage treatment for previously treated patients with chronic lymphocytic leukemia. PATIENTS AND METHODS: Fifty-two patients aged 39-84 years with previously treated CLL received cladribine 0.12 mg/kg/day in 2-hour infusions for 5 days in monthly courses. Two-thirds were refractory to previous therapy, and 8 had prior fludarabine. RESULTS: Sixteen (31%) patients achieved complete response (CR) and 14 (27%) partial remission (PR) according to consensus criteria. Response correlated with clinical stage, number of previous treatment regimes, blood lymphocyte count, and lymphocyte halflife following the first cladribine course. Toxicity included pneumonia (n = 9), herpes zoster (n = 7), and septicemia (n = 2). Four patients in CR underwent high-dose chemotherapy with autologous blood stem cell support, and 2 remain in CR 48 and 60 months from start of cladribine, and 2 had relapse at 42 and 48 months, respectively. Median progression-free survival (Kaplan-Meier analysis) for CR patients was 23 months from start of cladribine treatment, and for PR patients 16 months. The projected overall survival was 80% at 3 years for CR patients, and the median survival 28 months for PR patients and 4 months for non-responding patients. CONCLUSIONS: Our previous finding of durable CRs from cladribine in advanced CLL is thus confirmed in a larger patient material, and follow-up indicate that long-term survival may be achieved.     

The effects of patterned calorie-restricted diets on mammary tumor incidence and plasma endothelin levels in DMBA-treated rats

Chronic caloric restriction has been shown to inhibit mammary tumor promotion in the 7,12-dimethyl-benz[a]anthracene (DMBA) rat mammary tumor model. The objectives of this study were to determine (i) the effects of chronic caloric cycling (yo-yo dieting) on mammary tumor promotion by high fat diets and (ii) the effect of three dietary regimens +/- superimposed mammary tumor burden on plasma endothelin-1,2 (ET) levels. Female Sprague-Dawley rats were treated with DMBA (5 mg/rat) and divided into three dietary groups: ad libitum (AL) (containing 15% corn oil); 40% calorie restricted (CR) (containing 20% corn oil so consumption of fat was equivalent between AL and CR); a calorie cycled (CC) group fed alternatively AL and CR diets each 48 h period. After 10 weeks, tumor incidences were: AL, 63%; CR, 27%; CC, 57% (AL versus CR, P < 0.05; CC versus CR, P < 0.05; AL versus CC, NSD). ET levels (pg/ml plasma) were: AL, 16.0 +/- 6.54; CR, 32.31 +/- 0.34; CC, 23.44 +/- 5.04 (AL versus CR, P < 0.01; CC versus CR, P < 0.01; AL versus CC, P < 0.05). Plasma ET levels were independent of tumor incidence and tumor burden, but plasma ET levels were significantly increased in rats with a prior history of calorie restriction. As expected, maintained caloric restriction reduced mammary tumor incidence but intermittent caloric restriction (caloric cycling or yo-yo dieting) was without similar benefit.     

Boundary layer drug delivery using a helical catheter

BACKGROUND: A catheter-based approach for local endovascular drug delivery has been developed. The catheter is deployed percutaneously, while the end of the catheter is in the form of a helix that is placed just proximal to the vascular site to be treated. The helices are in contact with the vessel wall. A number of small holes is drilled in the coils of the catheter through which drug is infused, so that the infused drug remains within the blood fluid 'boundary layer' adjacent to the vessel wall. This approach is expected to be highly efficient for administration of antithrombotic and antiproliferative agents that target processes leading to vascular occlusion, heart attacks, and strokes. METHODS: The helical catheter was qualitatively evaluated using optical dye density measurements of Evans blue dye infused using an in vitro steady flow system under a physiologic range of conditions. To further demonstrate the efficiency of the technique, its capacity to inhibit thrombosis was evaluated in a baboon thrombosis model. The catheter was inserted into a femoral arteriovenous shunt (blood flow rate = 100 ml/min) and placed proximal to a segment of highly thrombogenic Dacron vascular graft (4.0 mm i.d.). Integrelin (an inhibitor of platelet glycoprotein IIb/IIIa; doses: 0.25-1.0 microg/min) and hirudin (an antithrombin; doses: 10-100 microg/min) were used to inhibit thrombus formation. RESULTS: Experimental flow visualization studies demonstrated that high concentrations of the infused Evans blue dye were retained near the vessel wall. In the animal experiments, platelet deposition on the Dacron graft surface was reduced by 82-97% (Integrelin) and 68-92% (hirudin) over 1-2 h of blood exposure. The local antithrombotic effects produced were found to be 200-fold and 30-fold more efficient than systemic administration of the same agents. CONCLUSIONS: Local drug infusion using the helical catheter approach can achieve high drug concentration levels at target sites, may avoid systemic effects, and can reduce cost of therapy by reducing total drug requirements.     

Meshlaparostomy--a comparison between polypropylene mesh and indigenous nylon net

Polypropylene mesh, the routinely used material for mesh laparostomies is expensive. A economical and easily available substitute-nylon net was compared with polypropylene mesh with regard to efficacy and wound complications in a retrospective study. 51 patients, 25 in the polypropylene mesh group and 26 in the nylon net group were studied. The groups were matched with respect to age, sex, indication for surgery and mortality. The mean hospital stay (65 vs 54 days), fate of the wound, incidence of mesh extrusion (26% vs 20%), bowel perforation (1 vs none) and incisional hernia formation (21% vs 20%) in those with polypropylene mesh and nylon net respectively was similar. In conclusion, nylon net is an effective and inexpensive indigenous substitute for polypropylene mesh for mesh laparostomies.