Chicken NGF and non-NGF trophic factor synthesis and release by sciatic nerves in vitro

The time course of production and release of nerve growth factor (NGF) and non-NGF neuronotrophic factors for sympathetic neurons by chicken and rat sciatic nerves in culture was examined. These tissues actively synthesize and release neuronotrophic activity as metabolically poisoning nerves with azide dramatically reduced the amount of trophic activity released into the culture medium. The sustained release of this activity also was shown to be dependent on the presence of low-molecular-weight dialysable molecules present in foetal calf serum and amniotic fluid from day 11 chicken embryos. Affinity-purified antimouse NGF antibodies were used to show that sciatic nerves in culture release both NGF and non-NGF trophic factors. These antibodies inhibited all bioactivity of both mouse NGF and of a partially purified preparation of chicken NGF. Immunoblot studies confirm that the antibodies recognize both rodent and avian NGF. Excess antibody inhibited only about 50% of the trophic activity in media conditioned over rat or chicken nerves for the first 24 hr. Relatively similar amounts of this non-NGF trophic activity were released throughout 6 days in culture, and this trophic activity kept sympathetic neurons alive in culture in the absence of NGF for more than 4 days. NGF levels were quantified with a two-site enzyme-linked immunoassay and found to parallel changes in NGF bioactivity. Rat nerves released increasing amounts of NGF with time in culture. Whole chicken sciatic nerves, however, released decreasing amounts of NGF with time in culture, but when these nerves were desheathed by removal of the epineurium and attached tissue, the pattern of NGF release was similar to that observed in the smaller rat sciatic nerves. These studies therefore characterize antibodies recognizing chicken NGF, demonstrate that peripheral nerve tissue synthesize trophic factors other than NGF, and identify factors that influence NGF synthesis.     

Localization of constitutive and hyperthermia-inducible heat shock mRNAS (hsc70 and hsp70) in the rabbit cerebellum and brainstem by non-radioactive in situ hybridization

Neural expression of constitutive hsc70 mRNA and hyperthermia-inducible hsp70 mRNA is examined using radioactive and non-radioactive in situ hybridization procedures. A strong induction of hsp70 mRNA was noted in cell populations in cerebellar layers and in the brainstem which demonstrated expression of mRNA encoding proteolipid protein, an oligodendrocyte marker. The non-radioactive in situ hybridization procedure using digoxigenin (DIG)-UTP-labeled riboprobes permitted improved signal localization, and stress-inducible hsp70 mRNA was detected at the cytoplasmic cap areas of individual oligodendrocytes. Cell types which express constitutive members of the hsc/hsp70 multigene family were also identified. Neurons in the brainstem and in the deep white matter and molecular layer of the cerebellum showed expression of hsc70 mRNA while signal was not detected in adjacent glial cells. A neuron-specific enolase riboprobe aided in the identification of neuronal cell types. The non-radioactive DIG riboprobe revealed that hsc70 mRNA was highly localized to the cyto-plasm of individual neurons. High constitutive levels of hsc70 in certain neurons may dampen hsp70 induction after hyperthermia in these cell populations. © 1995 Wiley-Liss, Inc.     

Salvage of the neuropathic foot by using bone resection and tendon balancing: A retrospective review of 10 patients

Medical records were retrospectively reviewed for 10 patients (mean age, 48.7 years) who had a chronic, recurrent neuropathic forefoot ulceration or osteomyelitis in the presence of an abnormal metatarsal parabola. Two patients had multiple lesser metatarsal osteomyelitis, 3 patients had chronic ulceration in the presence of an abnormal metatarsal parabola, and 5 patients had previous lesser ray resection or metatarsal head resection. None of the patients had signs of skin breakdown under the first metatarsal. All of the patients were treated with a combination gastrocnemius recession, peroneus longus to peroneus brevis tendon transfer, and resection of the second through fifth metatarsal heads to decrease plantar forefoot pressure and preserve the first ray without increasing the risk of ulceration under the first metatarsal head. All patients achieved a healed plantigrade foot without ulcer recurrence, transfer callus development, or contralateral foot breakdown at a mean follow-up of 14.2 months. Postsurgical complications consisted of dehiscence of various incision sites on 3 individual patients and one local reaction to antibiotic-impregnated beads. This preliminary study suggests that this combination of reconstructive procedures may provide an alternative method of foot salvage to panmetatarsal resection and transmetatarsal amputation.     

A probable waterborne outbreak of cryptosporidiosis in the Sheffield area

There was a marked peak in human cases of cryptosporidiosis in the Sheffield area in May and June 1986. Extensive epidemiological investigations failed to find a common source of food or a consistent history of animal contact, but did suggest that a waterborne outbreak of cryptosporidiosis may have occurred. Cryptosporidium oocysts were found in untreated water and in fish from a reservoir complex implicated by epidemiological analysis. Laboratory investigations confirmed that cattle on a farm adjacent to the reservoir complex were a possible source of contamination.     

Pretreatment with Isradipine, a Calcium-Channel Blocker, Does Not Attenuate the Acute Behavioral Effects of Ethanol in Humans

The acute subject-rated, performance-impairing, and physiological effects of ethanol (0, 0.5, and 1 g/kg) were examined after pretreatment with isradipine (0, 5, and 10 mg) in nine healthy volunteers. Volunteers received 1 of the 9 ethanol-isradipine combinations during each of nine experimental sessions. Ethanol alone produced prototypical subject-rated drug effects (e.g., increased ratings of "Drunk,""Good effects," and "Like drug") and impaired performance. Isradipine alone also produced significant subject-rated drug effects (e.g., increased ratings of "Drug effect,""Bad effects,""High," and "Stimulated"), but did not impair performance. Isradipine pretreatment generally did not significantly alter the subject-rated or performance-impairing effects of ethanol. Isradipine alone, but not ethanol alone, significantly decreased systolic and diastolic blood pressure. The ethanol-isradipine combinations generally produced significantly greater decreases in blood pressure than were observed with isradipine alone. Breath-alcohol levels were significantly lower after isradipine pretreatment, which suggests isradipine altered the bioavailability of ethanol. The present findings extend previous studies with humans that examined the behavioral effects of ethanol after pretreatment with other calcium-channel blockers, including nifedipine, nimodipine, and verapamil. Whereas the available studies suggest that calcium-channel blockers would not be useful pharmacological adjuncts in the management of ethanol abuse, more research is needed. Future studies should use self-administration and drug discrimination procedures adapted for use with humans to determine if calcium-channel blockers can attenuate any of the behavioral effects of ethanol.     

Modulation of the activity and assessment of the receptor selectivity in a series of new RGD-containing peptides

We have investigated the structure-activity relationship of a series of new synthetic RGD analogs and their potential use as specific platelet aggregation inhibitors. Twelve short linear peptides showed high potency to inhibit aggregation in ADP-stimulated dog platelets. In order to assess the selectivity of these analogs towards platelet integrin GPIIb-IIIa, a new cell adhesion inhibition system was devised which was able to discriminate between the two closely related β3-integrins of the vasculature, GPIIb-IIIa (α_IIbβ₃), present in platelets, and the vitronectin receptor (α_vβ₃), expressed in endothelial cells and platelets. As reported for other peptides by Scarborough et. al. (1993, J. Biol. Chem. 2 68 , 1066), the analogs containing lysine instead of arginine in position 1 showed increased selectivity towards GPIIb-IIIa. One of them, in which the piperidine carboxylic group was attached to the ^N-terminus of KGDW, not only strongly inhibited platelet aggregation, but also selectively abolished cell adhesion mediated by GPIIb-IIIa without effect on the vitronectin receptor.     

Long-Term Deterioration of Kidney Allograft Function

Although long-term survival after kidney transplantation is critically dependent on maintaining stable allograft function, few studies have examined renal allograft function over time. Using pooled data from 10 278 consecutive transplants at five centers, we calculated slopes of estimated glomerular filtration rates (eGFR) measured after 1, 6 and 12 months in 9515, 8861 and 7359 patients surviving > or =1, > or =6 and > or =12 months, respectively. Slopes of eGFR progressively diminished for patients transplanted during 1984-1989, 1990-1993, 1994-1998 and 1999-2002 (analysis of variance p < 0.0001 and p = 0.1245 for slopes measured after 1 and 6 months, respectively). Slopes measured after 12 months were less in the most recent era: -2.2 +/- 7.2 mL/min/1.73 m(2)/year, -2.3 +/- 6.6 mL/min/1.73 m(2)/year, -2.4 +/- 7.4 mL/min/1.73 m(2)/year and -1.4 +/- 10.9 mL/min/1.73 m(2)/year, respectively, p = 0.0058. Slopes measured after 1, 6 and 12 months each were less for transplantations during 1999-2002, after adjusting for multiple transplantation characteristics (p < 0.0001). Similarly, in Cox proportional hazards analysis, the risk (95% CI) for a 25% reduction in eGFR was 0.92 (0.85-1.01), p = 0.0736 during 1990-1994; 0.94 (0.82-1.08), p = 0.4111 during 1995-1998 and 0.78 (0.64-0.95), p = 0.0110 during 1999-2002 (compared to 1984-1989). We conclude that the rate of decline in allograft function after kidney transplantation has improved, suggesting that stable, long-term function may be achievable.     

Mechanism of toxicity of an experimental bidentate phosphine gold complexed antineoplastic agent in isolated rat hepatocytes

SK&F 104524 (bis-[1,2 bis(diphenylphosphino)-ethane]gold(l) lactate) [( Au(dppe)2]+) is an experimental antineoplastic agent that is hepatotoxic in vivo in the dog as well as highly cytotoxic to isolated canine hepatocytes in vitro. Preliminary studies in isolated dog hepatocytes have indicated that [Au(dppe)2]+ causes an increase in hepatocyte respiration and a decrease in cellular ATP. The purpose of the present investigation was to characterize [Au(dppe)2]+-induced cytotoxicity and biochemical lesions in the intact cell and to correlate these changes with mitochondrial function. The uptake of [14C][Au(dppe)2]+ by rat hepatocytes was rapid, reaching a maximum by 30 min. [Au(dppe)2]+ was distributed throughout the hepatocyte and associated rapidly with mitochondria, nuclei, cytosol and cellular membranes. [Au(dppe)2]+ caused cell lethality in a concentration-dependent fashion; although 5 microM did not cause any changes in lactic dehydrogenase leakage, 20 microM produced 100% cell death by 120 min. [Au(dppe)2]+ also caused concentration-dependent bleb formation of the hepatocyte plasma membrane, increased oxygen consumption and loss of ATP within 30 min. ATP loss was associated with transient increases in AMP and ADP and a profound drop in the ATP/ADP ratio and energy charge. Total nucleotides (adenine and xanthine nucleotides) remained constant. The pattern of glutathione depletion coincided with that of lactic dehydrogenase leakage. Electron microscopy of hepatocytes exposed to [Au(dppe)2]+ for 30 min revealed depletion of glycogen granules and marked swelling of mitochondria. In isolated rat liver mitochondria, [Au(dppe)2]+ caused a stimulation of state 4 respiration and loss of the respiratory control ratio. [Au(dppe)2]+ also relieved the oligomycin-induced inhibition of state 3 (ADP-stimulated) respiration.(ABSTRACT TRUNCATED AT 250 WORDS)