Modification of hypoxia-induced injury in cultured rat astrocytes by high levels of glucose

BACKGROUND AND PURPOSE: Preexisting hyperglycemia exacerbates central nervous system injury after transient global and focal cerebral ischemia. Increased anaerobic metabolism with resultant lactic acidosis has been shown to cause the hyperglycemic, neuronal injury. The contribution of astrocytes in producing lactic acidosis under hyperglycemic/ischemic conditions is unclear, whereas the protective role of astrocytes in ischemic-induced neuronal injury has been documented. The ability of astrocytes to maintain energy status and ion homeostasis under hyperglycemic conditions could ultimately reduce neuronal injury. Therefore, we determined the effects of increased glucose concentrations on glucose utilization, lactate production, extracellular pH, and adenosine triphosphate concentrations in hypoxia-treated astrocyte cultures. METHODS: Primary astrocytes were prepared from neonatal rat cerebral cortices. After 35 days in vitro, cultures were incubated with 0-60 mmol/L glucose and subjected to hypoxic conditions at 95% N2/5% CO2 for 24 hours. In addition, under high-glucose conditions (30 mmol/L), astrocytes were exposed to up to 72 hours of hypoxia. Determination of lactate dehydrogenase efflux, adenosine triphosphate concentrations, and extracellular lactate concentrations defined astrocyte status. Equiosmolar levels of mannitol were added in place of high glucose concentrations to distinguish hyperosmotic effect. RESULTS: When physiological concentrations of glucose (7.5 mmol/L) or lower concentrations were used, significant cell damage occurred with 24 hours of hypoxia, as determined by increased efflux of lactate dehydrogenase and loss of cell protein. When higher glucose concentrations (15-60 mmol/L) were used, efflux of lactate dehydrogenase was similar to that observed in normoxic cultures, despite an increased utilization of glucose. Lactate concentrations in the media at low or normal glucose concentrations exceeded normoxic levels, but higher glucose concentrations (15-30 mmol/L) failed to increase lactate levels further. Values of adenosine triphosphate for hypoxic astrocytes treated with high glucose concentrations were significantly higher than those of astrocytes with zero or low glucose levels. In cultures exposed to hypoxia and high glucose levels (30 mmol/L), no cellular injury was observed before 48 hours of hypoxia. Lactate concentrations in the media increased during the first 24 hours of hypoxia and reached steady state. The pH of the media decreased to 6.4 after 24 hours and 5.5 at 48 hours. The latter pH was concomitant with a marked increase in extracellular lactate dehydrogenase activity. Hyperosmotic mannitol failed to protect cultured astrocytes against hypoxia. CONCLUSIONS: Hypoxic injury to mature astrocytes was reduced by the presence of 15-60 mmol/L glucose in the medium during 24-30 hours of hypoxia. Injury occurred when the pH of the medium was < 5.5. This protection was not afforded by the hyperosmotic effect of high glucose concentrations, nor was the hypoxic injury at later time periods with 30 mmol/L glucose mediated solely by lactate accumulation.     

The Glu318Gly mutation of the presenilin-1 gene does not necessarily cause Alzheimer's disease

In early-onset familial Alzheimer's disease (AD) pathogenic mutations have been found in the amyloid precursor protein (APP) gene and in the presenilin (PS)-1 and PS-2 genes. We screened for mutations in these genes in 20 patients with familial AD from the Finnish population. In addition, we sampled 41 sporadic AD patients and 59 controls to test for mutations identified in our familial AD cases. We detected an A-to-G transition in the PS-1 gene, resulting in a glutamic acid (Glu)-to-glycine (Gly) substitution at codon 318 in 2 familial and 2 sporadic AD patients. The Glu318Gly mutation has previously been reported to cause AD. We also found the Glu318Gly mutation in 4 healthy aged controls (range, 74-87 years). We thus conclude that the mutation is most likely a rare polymorphism not related to AD.     

Ultrahigh Vacuum Scanning Tunneling Microscope-Based Nanolithography and Selective Chemistry on Silicon Surfaces

Nanofabrication on silicon surfaces has been achieved in a manner similar to e-beam/resist technology, in which hydrogen serves as a monolayer resist for exposure by the electron beam from an ultrahigh vacuum (UHV) scanning tunneling microscope (STM). In this scheme, hydrogen is selectively desorbed from Si(100)2×1:H surfaces that have been prepared by atomic hydrogen dosing under UHV background conditions. To remove hydrogen, the tip bias is raised, under feedback control, and then the desired pattern is drawn. Two regimes of hydrogen desorption are observed: at higher energies, above ∼6.0 V, direct electron-stimulated desorption occurs, whereas at lower biases, desorption occurs via a multiple excitation vibrational heating mechanism and exhibits a strong current dependence. Patterning linewidth down to a single dimer row has been achieved in the vibrational heating regime. The selective removal of hydrogen suggests many possibilities for subsequent chemical treatments in which the hydrogen-terminated silicon remains inert. We have performed experiments which demonstrate selective oxidation of, and nitrogen incorporation into, the STM-patterned regions.     

Neuronal spike activity in the nucleus accumbens of behaving rats during ethanol self-administration

Many lines of evidence support the importance of the nucleus accumbens (NAC) for ethanol-reinforced behavior. The nature of the neuronal activity that occurs in this region during ethanol self-administration is not known. We recorded from ensembles of single-units primarily located within the shell of the NAC during operant responding for oral ethanol solutions by well-trained rats. Of 90 units recorded from seven sessions from seven rats, 41 (46%) did not exhibit significant changes in relation to the experimental events. Of the 49 units (54%) that did exhibit significant phasic changes, alterations in firing rate occurred in relation to the following experimental events: operant response (63%), tone stimulus (20%), and ethanol delivery (63%). In addition, changes in spike activity during the intervals between the three experimental events were noted in 33% of the units. Most units (55% of responsive units) responded to multiple experimental events. Thus different but overlapping populations of neurons in the NAC represent each event that occurs along the temporal dimension of a single trial performed to obtain ethanol reward. The data suggest that the NAC plays a crucial role in linking together conditioned and unconditioned internal and external stimuli with motor plans to allow for ethanol-seeking behavior to occur.     

Biosynthesis of scarab beetle pheromones

Chemical communication in scarab beetles (Coleoptera: Scarabaeidae) is achieved with a wide variety of pheromones, but one typical structure is the gamma-lactone having a long unsaturated hydrocarbon chain. Several species utilize (R, Z)-5-(-)-(oct-1-enyl)-oxacyclopentan-2-one (buibuilactone), (R, Z)-5-(-)-(dec-1-enyl)-oxacyclopentan-2-one and (S, Z)-5-(+)-(dec-1-enyl)-oxacyclopentan-2-one [(R)-japonilure and (S)-japonilure]. Using deuterated precursors, we have demonstrated that these compounds are biosynthesized from fatty acids. (9, 10-d4)-Palmitic acid, (9,10-d4)-stearic acid, (9,10-d2)-palmitoleic acid, (9,10-d2)-oleic acid, (9,10-d2)-8-hydroxypalmitoleic acid and (9,10-d2)-8-hydroxyoleic acid were readily incorporated by female Anomala cuprea into the pheromone molecules, while (Z)-(5, 6-d2)-5-dodecenoic acid and (Z)-(5,6-d2)-5-tetradecenoic acid were not. Therefore, the reaction pathway starts from saturated fatty acids, involves their desaturation, followed by 8-hydroxylation, chain shortening and cyclization. The products obtained from racemic (9,10-d2)-8-hydroxypalmitoleic acid and (9,10-d2)-8-hydroxyoleic acid were also racemic, implying that the steps following hydroxylation were not stereospecific. Perdeuterated palmitic acid was applied to disclose the mechanism of the unique hydroxylation reaction. Retention of all deuterium atoms implied that this reaction was a direct process mediated by a specific fatty acid hydroxylase, and preceding desaturation or epoxidation was not involved.     

A combined approach of conventional and molecular cytogenetics for detailed karyotypic analysis of the small cell lung carcinoma cell line U2020

Until recently the ability to analyze complex karyotypic rearrangements was totally dependent upon light microscopy of G-banded chromosomes. Developments in the area of molecular cytogenetics have revolutionized such analysis, making it possible to determine the nature of complex rearrangements. An extensive analysis has been made of the small cell lung carcinoma (SCLC) cell line U2020, using a combined approach of conventional and molecular cytogenetics, enabling a highly detailed karyotype to be constructed revealing rearrangements previously undetected by G-banding alone. This approach offers the opportunity to reassess other tumor karyotypes, particularly those of high complexity found in solid tumors, for tumor-specific consistent rearrangements indecipherable by conventional karyotyping.     

Reduction of apurinic/apyrimidinic endonuclease expression after transient global cerebral ischemia in rats: implication of the failure of DNA repair in neuronal apoptosis

BACKGROUND AND PURPOSE: To clarify the relationship between apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, and delayed neuronal cell death associated with apoptosis, we examined the expression of APE/Ref-1 before and after transient global ischemia in rats. METHODS: Global ischemia was induced by bilateral common carotid artery occlusion and hypotension. Expression of the APE/Ref-1 protein was evaluated by Western blot and immunohistochemical analyses. Apoptosis after global ischemia was observed by DNA electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. RESULTS: Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the control brains. Nuclear immunoreactivity of APE/Ref-1 was significantly decreased 2 days after 10 minutes of ischemia in the hippocampal CA1 subregion. Western blot analysis of a sample from the normal brains showed a characteristic 37-kDa band, which was reduced in the hippocampal CA1 subregion after ischemia. A significant amount of DNA fragmentation was observed at 3 days but not at 1 day after ischemia. Double staining with APE/Ref-1 and TUNEL clearly showed that the neurons that lost APE/Ref-1 immunoreactivity became TUNEL positive. CONCLUSIONS: Our data provide evidence that APE/Ref-1 decreased in hippocampal CA1 neurons after transient global ischemia and that this reduction precedes DNA fragmentation, which is destined to cause apoptosis. Our results suggest the possibility that a decrease of APE/Ref-1 activity and the failure of DNA repair may underlie the mechanism of apoptosis after transient focal ischemia.     

Early detection of recurrent prostate cancer with an ultrasensitive chemiluminescent prostate-specific antigen assay

OBJECTIVES: Treatment failure after radical prostatectomy is most commonly heralded by an increase in serum prostate-specific antigen (PSA) to detectable levels. We evaluated the clinical utility of an ultrasensitive chemiluminescent PSA assay. METHODS: We evaluated the assay in banked sera obtained from 170 men after radical prostatectomy. Controls consisted of 142 females, 29 men who had undergone cystoprostatectomy without evidence of prostate cancer, and 25 men without evidence of recurrent disease at least 5 years after prostatectomy for organ-confined disease. Lead time to diagnosis of recurrence was based on comparisons with the IMx or Tandem E assays using a cutoff of 0.1 ng/mL (100 pg/mL). RESULTS: The biologic level of detection of this assay is 8 pg/mL. Serum PSA levels were undetectable in 82.4% of females, 86.2% of the cystoprostatectomy patients, and 96% of the radical prostatectomy controls. After radical prostatectomy, PSA levels were undetectable at last check in 104 of 168 (61.9%) men. In the 24 men with prostate cancer recurrence, the enhanced sensitivity of 8 pg/mL provided a mean lead time based on conservative calculations of 12.7 to 22.5 months over conventional assays. Thirty-four of the 41 men with detectable PSA levels and no evidence of disease recurrence had PSA levels of 30 pg/mL or less. CONCLUSIONS: PSA levels are undetectable in most men who do not have recurrence of disease after radical prostatectomy. Low but detectable serum PSA levels less than or equal to 30 pg/mL can be produced by nonmalignant sources of PSA. PSA assays with enhanced sensitivity can detect recurrent prostate cancer with significant lead time over conventional assays.