Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein.

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed.

Augmenter of liver regeneration enhances the success rate of fetal pancreas transplantation in rodents.

BACKGROUND: Treatment of fetal pancreas (FP) isografts with insulin-like growth factor-I greatly improves the rate of conversion to euglycemia in diabetic rats. Complete knowledge of other factors that may facilitate the engraftment and function of FP in vivo is still embryonic. Augmenter of liver regeneration (ALR) is a newly described polypeptide growth factor found in weanling rat livers. ALR has trophic effects on regenerating liver. We studied the effects of in situ administration of this agent on FP isografts in rats. METHODS: Streptozotocin-diabetic Lewis rats (blood glucose > 300 mg/dl) received 16 FP isografts transplanted intramuscularly. ALR was delivered from day 1 through day 14, in doses of 40 or 400 ng/kg/d. Animals were followed for 3 months with serial weights and blood glucose monitoring. These animals were compared with those treated with vehicle alone. RESULTS: Of the group treated with ALR at 40 ng/kg/day for 14 days, 89% (eight of nine) were euglycemic (P=0.0003). Of the group treated with ALR at 400 ng/kg/day for 14 days, 88% (seven of eight) were euglycemic (P=0.0007). Of the group treated with vehicle alone, none of the six were euglycemic. Euglycemia is defined here as glucose < 200 mg/dl for 3 days. Pathology of the intramuscular transplant site showed patches of islet tissue embedded in fat. These patches demonstrated insulin immunoreactivity. CONCLUSIONS: Diabetes was reversed in a significantly greater proportion of FP + ALR-treated recipients than those animals treated with vehicle alone. Local delivery of growth factors may be used as an adjunct to FP transplantation to improve the rate of success. This in situ model may be useful to further evaluate other soluble factors.

The influence of combined trophic factors on the success of fetal pancreas grafts.

BACKGROUND: Fetal pancreas (FP) has the capacity for abundant proliferation and beta cell differentiation. Insulin-like growth factor-1 (IGF-1) promotes FP engraftment in the i.m. site and reversal of diabetes in a rodent model. However, reversal of diabetes by an FP transplant in rats under the influence of IGF-1 is still an inefficient process requiring multiple FP grafts and a prolonged latent period. Numerous other growth and differentiation factors, which include platelet derived growth factor (PDGF), vascular endothelial growth factor, endothelial cell growth factor-alpha and pancreatic islet neogenesis-associated protein, have been implicated in beta cell neogenesis and proliferation. We have analyzed the in vivo role of these growth factors in FP engraftment and reversal of streptozotocin-induced diabetes in rats. METHODS: IGF-1 alone or in combination with other trophic factors was locally administered to eight FP isografts in the thigh muscle of diabetic rats. RESULTS: Diabetes was reversed in a mean of 60+/-26 days in 11 of 11 animals treated with IGF-1. PDGF alone did not promote reversal of diabetes; however, PDGF + IGF-1 resulted in euglycemia in 6 of 6, with a mean of 36+/-14 days (P<0.05). Islet neogenesis-associated protein +IGF-1 resulted in reversal of diabetes in 6 of 6 rats with a mean interval of 50+/-10 days. Vascular endothelial growth factor or endothelial cell growth factor-alpha + IGF-1 provided no advantage compared with IGF-1 alone. CONCLUSIONS: These results demonstrate that IGF-1 is a potent trophic factor for transplanted FP and that PDGF acts synergistically with IGF-1 to promote reversal of diabetes by transplanting FP.

Insulin-like growth factor-I ameliorates delayed kidney graft function and the acute nephrotoxic effects of cyclosporine.

BACKGROUND: Delayed graft function (DGF) is a relatively common complication after cadaveric renal transplantation. The adverse effect of DGF on long-term graft survival has lead to intensive efforts to reduce ischemic graft injury. In this study we examined the effects of a new protective treatment based on insulin growth factor (IGF)-I. We evaluated the impact of the treatment on renal recovery and on the nephrotoxicity that is a common side effect of mainstream immunosuppressants. Because therapy with IGF-I or the analog des(1-3)IGF-I is effective in treating experimental ischemic renal failure, these peptides may be useful as perspective clinical treatments. METHODS: We have addressed three areas relating to the potential use of IGF-I and its analog des(1-3)IGF-I. First, because of the immunogenic properties of IGF-I, we assessed the effect of des(1-3)IGF-I on the rejection of skin allografts in Lewis rats. Next we determined whether treatment with des(1-3)IGF-I influences the early function of transplanted kidneys in a model of DGF induced by a combination of warm and cold ischemia. Finally we tested whether IGF-I protects against acute cyclosporine nephrotoxicity. RESULTS: Des(1-3)IGF-I did not accelerate the rejection of the skin grafts (P=0.57). The administration of this peptide in a model of syngenic renal transplant improved the early function of the graft. Postoperative values of creatinine and blood urea nitrogen were significantly better (P<0.05) in treated animals. IGF-I also ameliorated the nephrotoxicity of cyclosporine, with better values of creatinine and blood urea nitrogen (P<0.05). CONCLUSIONS: In evaluating this study it should be recognized that the animal models studied, although widely used, differ from the human condition. However, IGF-I and des(1-3)IGF-I exhibit properties that strongly suggest their value in preventing clinical DGF, and they deserve further studies.

Platelet accumulation on collagen: drugs which inhibit arachidonic acid metabolism and affect intracellular cyclic AMP levels.

We have studied the accumulation of washed platelets on collagen-coated glass from flowing platelet-red blood cell suspensions in the presence and absence of drugs. Glass tubes were 10 cm long and the flow rate was 1 ml/min, 80 s-1. For all experiments, platelet accumulation was greatest near the tube's inlet with a continuous decrease to the exit. A common feature, of those drug treatments which lead to reduced accumulation at the inlet, was an increase in outlet accumulation when compared with outlet control values. Platelet-collagen adhesion resulted in maximal release of 3H-serotonin in the presence of agents that prevent platelet aggregation on collagen. Only drug treatment known to raise cAMP levels (PGE1 and dipyridamole) or prevent the formation of prostaglandins and thromboxanes (sulfinpyrazone, indomethacin and ASA) were found to inhibit aggregate growth. Platelet aggregation on collagen in the absence of thrombin likely proceeds through the liberation of prostaglandins and thromboxanes from surface-bound platelets into the flow where they may stimulate flow-born cells. An alternate hypothesis is that such treatments affect the release of alpha-granule components necessary for aggregation.

Inhibition of human platelet phospholipase A2 by mono(2-ethylhexyl)phthalate.

There is evidence that the carcinogenic and teratogenic effects attributed to the plasticizer di(2-ethylhexyl)phthalate (DEHP) are due to its major metabolite mono(2-ethylhexyl)phthalate (MEHP). MEHP is also formed ex vivo by a plasma enzyme in blood products stored in polyvinyl chloride (PVC) DEHP plastic containers. People who receive large amounts of blood products, such as hemophiliacs or patients undergoing hemodialysis, cardiopulmonary bypass, or massive transfusion, are exposed to significant levels of plasticizer. In this study, the platelet was used to show that MEHP inhibits phospholipase A2 (PLA2), one of enzymes important in the release of arachidonic acid from membrane phospholipids. Arachidonate is the parent molecule for the synthesis of prostaglandins, thromboxanes, leukotrienes, and lipoxins that are made by a wide variety of cells. PLA2 was measured by the liberation of 14C-arachidonic acid from 1-stearoyl-2-[1-14C]arachidonyl-L-3-phosphatidylcholine. MEHP inhibits PLA2 activity noncompetitively in intact human platelets and lysates with a Ki of 3.7 x 10(-4) M. DEHP does not inhibit PLA2 in whole platelets. Inhibition of PLA2 by MEHP occurs at only three times the circulating level of MEHP measured in neonates undergoing exchange transfusion and 20-fold the levels experienced by patients during cardiopulmonary bypass. Therefore, infants and adult patients with multisystem failure who accumulate MEHP in their blood may be at risk for decreased platelet function.

Insulin-like growth factor-I promotes successful fetal pancreas transplantation in the intramuscular site.

BACKGROUND: Fetal pancreas (FP) has the capacity for exuberant proliferation and engrafts in the safe, accessible intramuscular site. These characteristics make FP transplantation an attractive approach to the treatment of diabetes mellitus. Insulin-like growth factor-I (IGF-I) is an important mediator of growth and maturation of many tissues and has been shown to induce fetal islet proliferation. METHODS: IGF-I was locally administered at a rate of 69 micrograms/kg/day to 0, 2, 4, 8, or 16 FP isografts placed into the thigh muscle of streptozotocin-induced diabetic Lewis rats (blood glucose > 350 mg/dl). RESULTS: Diabetes was reversed in eight of eight animals receiving 16 FP and treated with IGF-I. One of seven animals receiving 16 FP without IGF-I treatment became euglycemic (p = 0.003). Similar improved conversion rates were seen in groups of animals receiving either eight, four, or two FP and treated with IGF-I, compared to groups receiving eight, four, or two FP without IGF-I treatment. Euglycemic recipients had physiologic glucose tolerance. The interval to conversion increased inversely in proportion to the amount of FP tissue transplanted. CONCLUSIONS: These results show that local delivery of IGF-I has potent trophic effects on FP transplanted to the intramuscular site.

Resilience-recovery factors in post-traumatic stress disorder among female and male Vietnam veterans: hardiness, postwar social support, and additional stressful life events.

Structural equation modeling procedures were used to examine relationships among several war zone stressor dimensions, resilience-recovery factors, and post-traumatic stress disorder symptoms in a national sample of 1,632 Vietnam veterans (26% women and 74% men). A 9-factor measurement model was specified on a mixed-gender subsample of the data and then replicated on separate subsamples of female and male veterans. For both genders, the structural models supported strong mediation effects for the intrapersonal resource characteristic of hardiness, postwar structural and functional social support, and additional negative life events in the postwar period. Support for moderator effects or buffering in terms of interactions between war zone stressor level and resilience-recovery factors was minimal.

A simplified approach to the analysis of subclasses of phospholipids: application to human platelets.

A procedure for the determination of the proportions of diacyl, alkenylacyl and alkylacyl subclasses of glycerophospholipids was developed. The procedure involves: (1) acid methanolysis of the phospholipid followed by Bligh/Dyer extraction of fatty acid methyl esters (FAME) derived from acyl chain types, dimethylacetals (DMA) derived from alkenyl ether chain types, and lysoalkyl phosphatidic acids (lysoalkyl-PA) derived from alkyl ether chain types; and (2) subsequent acetolysis to convert the lysoalkyl-PA to monoalkyl glycerol diacetates (MAGD). GLC analysis and quantitation (using internal standard, 21:0 FAME) of FAME, DMA and MAGD allowed calculation of the proportions of the three molecular subclasses. The methanolysis/acetolysis procedure gave an overall mean phospholipid recovery of 95 +/- 3%. Analysis of the major phospholipids in four separate preparations of fresh resting human platelets by this procedure showed the following range of molecular subclasses: phosphatidylcholine (PC), 86-92 mol % diacyl, 6-10 mol % alkylacyl and 2-3 mol % alkenylacyl; and phosphatidylethanoline (PE), 39-60 mol % diacyl, 5-8 mol % alkylacyl and 34-55 mol % alkenylacyl. The results of these subclass analyses were in general agreement with those reported in the literature.

Chemical synthesis and physiological activity of sulfonium analogues of platelet activating factor.

Phosphatidylsulfocholine (PSC), the sulfonium analogue of phosphatidylcholine (PC), occurs naturally in some diatoms. The replacement of the [formula; see text] group by a [formula; see text] results in an increase in the polar head group size in PSC relative to that of PC, consistent with the observed increase in permeability of PSC bilayers towards urea. It was of interest to see whether replacement of the [formula; see text] group in platelet activating factor (PAF) by an [formula; see text] group leads to any change in platelet aggregation or other physiological activity. Synthesis of the sulfonium analogue of PAF was carried out by suitable modifications of known procedures. The PAF-sulfonium analogue was found to have almost the same platelet aggregating activity as PAF itself, in the concentration range 1-20 microM, but a much lower activity in the range 0.01-1 microM. The analogue had little or no effect on the platelet aggregation activity of PAF when added in the concentration range 0.01-1 microM and had about half the hypotensive activity of PAF towards hypertensive CDF male rats. The sulfonium analogue, however, was much more cytotoxic to HL-60 cells than PAF itself, in the concentration range 0-15 microM; replacement of the acetate group by a benzyl group increased the cytotoxicity to the level of that of the methoxy analogue of PAF. Thus, replacement of the [formula; see text] group by a [formula; see text] group in the polar head group region of PAF results in a relatively small change in its platelet aggregation activity and a decrease in its hypotensive activity, but greatly increases its antitumor activity.

Thoracic neuroblastoma: a Pediatric Oncology Group study.

Ninety-six patients with thoracic neuroblastoma were studied in a prospective fashion. Median age at presentation was 0.9 years. Forty-eight percent of the patients presented with stage A disease, 20% stage B, 13% stage C, 17% stage D, and 2% stage DS. Seventy-five patients have been followed for greater than 4 years. A posterior mediastinal mass was diagnosed on incidental chest roentgenograms performed for nontumor-related symptoms in 49% of the cases. Sixteen percent of the cases presented with neurological symptoms and 14% of the patients presented with acute respiratory distress. Urinary catecholamines were elevated in 76% of the cases. Complete surgical resection was carried out in 47% of the cases, while incomplete resection or biopsy was performed in 45%. No operation was performed in 3 patients. Minor surgical complications occurred in 20% of the patients, and 3% of the patients had significant perioperative complications. One patient died as a complication of therapy. Overall actuarial survival was 88% at 4 years. This study confirms the favorable outcome in children with mediastinal neuroblastoma. The basic biology of thoracic neuroblastomas seems to differ from that of other sites in that the majority of patients present at a younger age with localized disease or regional lymph node metastases, and have an improved survival even after correcting for age and stage. While complete excision is recommended, if possible, radical surgical procedures are not indicated since an excellent prognosis is associated with combined modality therapy.

Relationships between time management, control, work-family conflict, and strain.

This article incorporates recent research regarding time management into a model of work-family conflict. The authors hypothesized that 3 types of time management behavior would have both direct and indirect (through perceived control of time) relationships, with work interfering with family and family interfering with work. It was also hypothesized that both of these types of work-family conflict would be related to the strain outcomes of job dissatisfaction and health complaints. This model was tested with a sample of 522 workers. In general, the hypothesized relationships were supported.

Storage of human platelet concentrates in an artificial medium without dextrose.

It was shown previously that human blood platelets stored in an artificial medium (PCD) for up to 5 days remain functional in vitro and have normal survival and recovery in vivo. This report demonstrates that the medium can be simplified further by the removal of dextrose, leaving for study a medium consisting simply of balanced salts and citrate anticoagulant (PC). Some dextrose, 3.2 mM, was present in the fresh PC platelet concentrates due to plasma carryover in the production of platelet concentrates, but this dextrose concentration was considerably less than the 22.6 to 25.5 mM present in platelet concentrates in PCD or plasma. Platelet count, pH, PCO2, and PO2, as well as platelet aggregation and release responses to stimulation, in vitro, were as well preserved in the PCD or PC media as in the plasma controls. In the PC medium, platelets consumed 2.5 mM dextrose over 5 days and left 0.7 mM dextrose. The same consumption of dextrose was noted in PCD platelet concentrates, while platelets in plasma metabolized twice as much dextrose and formed twice as much lactate. Thus, the rate of glycolysis in platelet concentrates was independent of the dextrose concentration in the medium, and the platelet functions were well preserved.