Codistribution of procathepsin B and mature cathepsin B forms in human prostate tumors detected by confocal and immunofluorescence microscopy

Ascorbic acid can recycle alpha-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle alpha-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized alpha-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of alpha-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of alpha-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte alpha-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of alpha-tocopherol from both cells and from alpha-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas alpha-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects alpha-tocopherol in the cell membrane by a direct recycling mechanism.     

Topical gentamicin and ethacrynic acid: effects on cochlear function

OBJECTIVE: To determine whether concurrent intravenous administration of the loop diuretic ethacrynic acid potentiates the toxicity of the aminoglycoside antibiotic gentamicin applied topically on the round window. STUDY DESIGN: The authors studied the effects on cochlear sensitivity of co-administered intracardiac ethacrynic acid (40 mg/kg) and high-dose topical gentamicin solution (100%) applied to the round window. Comparisons were made with animals receiving ethacrynic acid plus systemic gentamicin (100 mg/kg); topical gentamicin alone; systemic gentamicin alone; and intravenous ethacrynic acid alone. METHODS: Experiments were carried out on pigmented guinea pigs weighing 400 to 500 g. Changes in cochlear function were characterized by monitoring shifts in compound action potential (CAP) thresholds by use of chronic indwelling electrodes implanted at the round window, vertex, and contralateral mastoid. RESULTS: After 20 days animals receiving ethacrynic acid in combination with topical gentamicin to the round window failed to demonstrate a significant deterioration in cochlear sensitivity, whereas all animals receiving systemic gentamicin plus ethacrynic acid experienced profound increases in CAP thresholds. CONCLUSIONS: This study supports the contention that ethacrynic acid potentiates aminoglycoside ototoxicity by facilitating the entry of the antibiotics from the systemic circulation into the endolymph. In addition, this study answers important clinical concerns regarding the safety of the use of topical aminoglycoside agents in combination with loop diuretics.     

Pituitary adenylate cyclase-activating polypeptide stimulates both c-fos gene expression and cell survival in rat cerebellar granule neurons through activation of the protein kinase A pathway

A high density of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors coupled to both adenylyl cyclase and phospholipase C is found in the external granule cell layer of the rat cerebellum during postnatal development. It has recently been reported that synthetic PACAP promotes cell survival and neurite outgrowth in immature granule cells. In the present study, we have investigated the transduction pathways that mediate the neurotrophic activity of PACAP in cultured granule cells from eight-day-old rat cerebellum. The effect of PACAP on cell survival was mimicked by dibutyryladenosine 3',5'-cyclic-monophosphate but not phorbol 12-myristate 13-acetate suggesting that only the adenylyl cyclase pathway is involved in the neurotrophic activity of PACAP. PACAP also induced a transient increase in c-fos messenger RNA level. The ability of PACAP to stimulate c-fos gene expression was mimicked by dibutyryladenosine 3',5'-cyclic-monophosphate but not phorbol 12-myristate 13-acetate. Similar effects of PACAP on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The protein kinase A inhibitor H89 significantly reduced the effect of PACAP on c-fos messenger RNA level whereas the specific protein kinase C inhibitor chelerythrine did not modify c-fos gene expression. These data indicate that the action of PACAP on cerebellar granule cell survival and c-fos gene expression are both mediated through the adenylyl cyclase/protein kinase A pathway. The observation that a short-term stimulation by PACAP can be converted into a long-lasting response indicates that the effect of the peptide on cell survival must involve immediate-early gene activation. The fact that a brief exposure to PACAP causes both c-fos gene expression and promotes cell survival strongly suggests that c-fos is involved in the trophic effect of PACAP on immature cerebellar granule cells.     

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.     

Longitudinal assessment of inpatient use and functioning of seriously mentally ill veterans with and without co-occurring substance use disorders

Within a fungal species, a subset of individuals may have more than the minimal complement of chromosomes. If the extra chromosomes are composed primarily of DNA not found in all representatives of the species, they are most appropriately referred to as supernumerary chromosomes. The patterns of repeated DNA sequences on certain supernumerary chromosomes suggest that they have a different evolutionary history from the essential chromosomes in the same genome. Supernumerary chromosomes can carry functional genes and, in at least two fungal species, genes on such chromosomes play important roles in host-pathogen interactions. Supernumerary chromosomes that confer an adaptive advantage in certain habitats, such as the ability to cause disease on a specific host, may be referred to as "conditionally dispensable" chromosomes in order to reflect their importance in some, but not all, growth conditions. In addition to describing the structural and functional characteristics of known supernumerary chromosomes in fungi, this review discusses the relative merits of the terms that have been used to describe them, and establishes experimental criteria for their identification.     

The influence of DNA glycosylases on spontaneous mutation

In the present study we examined the effects of phasic activation of the nucleus locus coeruleus (LC) on transmission of somatosensory information to the rat cerebral cortex. The rationale for this investigation was based on earlier findings that local microiontophoretic application of the putative LC transmitter, norepinephrine (NE), had facilitating actions on cortical neuronal responses to excitatory and inhibitory synaptic stimuli and more recent microdialysis experiments that have demonstrated increases in cortical levels of NE following phasic or tonic activation of LC. Glass micropipets were used to record the extracellular activity of single neurons in the somatosensory cortex of halothane-anesthetized rats. Somatosensory afferent pathways were activated by threshold level mechanical stimulation of the glabrous skin on the contralateral forepaw. Poststimulus time histograms were used to quantitate cortical neuronal responses before and at various time intervals after preconditioning burst activation of the ipsilateral LC. Excitatory and postexcitatory inhibitory responses to forepaw stimulation were enhanced when preceded by phasic activation of LC at conditioning intervals of 200-500 ms. These effects were anatomically specific in that they were only observed upon stimulation of brainstem sites close to (>150 micron) or within LC and were pharmacologically specific in that they were not consistently observed in animals where the LC-NE system had been disrupted by 6-OHDA pretreatment. Overall, these data suggest that following phasic activation of the LC efferent system, the efficacy of signal transmission through sensory networks in mammalian brain is enhanced.     

Spinal injuries in New Zealand rugby and rugby league--a twenty year survey

AIMS: To establish trends in frequency of serious spinal cord injuries in rugby and rugby league over a 20 year period and to elucidate patterns of injury from retrospective analysis of cases admitted to New Zealand's two spinal injuries units. METHODS: A detailed survey of unit records with follow-up of selected patients; statistical analysis of data. RESULTS: During the 20 years 1976 to 1995, 119 rugby and 22 rugby league players (total 141) were admitted to New Zealand's two spinal injuries units suffering serious spinal injuries and 47 of these became permanently confined to wheelchairs. There was a steady increase in frequency throughout the period studied. Of the injuries 83% occurred in forwards and 17% in backs. In rugby it was the scrum which produced most injuries, and in rugby league it was the tackle. The early season month of April produced most spinal injuries. In the eighteen months since intense compulsory educational programmes on safety were introduced by the New Zealand Rugby Union there have been no serious spinal cord injuries from rugby scrums. CONCLUSION: Contrary to widespread belief, there has not been a decrease in spinal cord injuries in rugby following rule changes in the mid 1980s. The information produced by this retrospective study has been an effective educational platform to make rugby and rugby league safer.