Electrophysiological and pharmacological evidence in favor of amino acid neurotransmission in fimbria-fornix fibers innervating the lateral septal complex of rats

Summary Electrical stimulation of fimbria-fornix (fifx) fibers monosynaptically activated many of the neurons tested in the lateral septal complex (LSC) of the rat. The orthodromically activated LSC neurons were classified as strongly orthodromically activated (SOA) or weakly orthodromically activated (WOA) cells according to their threshold for eliciting a response, stability of the response latency, frequency following and the stimulus-response ratio.Microiontophoretically applied glutamate (GLU) could excite both SOA and WOA neurons. However, the expelling currents needed to activate the SOA cells were often considerably lower than those necessary to excite the WOA cells suggesting higher sensitivity to GLU of those cells which receive a strong fi-fx innervation. Iontophoretically administered glutamic acid diethylester (GDEE) in general reversibly attenuated excitatory responses of LSC cells to GLU but not to acetylcholine. GDEE was also effective in blocking the synaptic responses of SOA septal cells to fi-fx stimuli. In addition, GDEE administered topically reversibly suppressed the field potential induced in the LSC by fi-fx stimulation.These electrophysiological and pharmacological results support recent biochemical observations suggesting that the excitatory innervation of LSC neurons by fi-fx fibers is mediated by GLU or a closely related excitatory amino acid.The investigations were supported by the Foundation for Medical Research FUNGO which is subsidized by the Netherlands Organization for Advancement of Pure Research (Z.W.O.), grant 13-31-045 awarded to I.J. A. Urban     

Upregulation of the Golgi protein GP73 by adenovirus infection requires the E1A CtBP interaction domain

GP73 is a novel type II Golgi transmembrane protein that is expressed at high levels in the hepatocytes of patients with viral hepatitis (R. D. Kladney, G. A. Bulla, L. Guo, A. L. Mason, A. E. Tollefson, D. J. Simon, Z. Koutoubi, and C. J. Fimmel, 2000, Gene 249, 53-65) and is induced in cultured cells by infection with viruses including adenoviruses. Its biological function and the mechanisms by which its expression may be regulated by viral infection are unknown. Here we report that GP73 is induced at the RNA and protein level in human Hep3B hepatoma cells infected by human Ad5 and Ad2. Hep3B cells were infected with wild-type or mutant adenoviruses. GP73 expression was measured by RNase protection assay, immunoblotting, or immunofluorescence microscopy. GP73 RNA and protein levels were strikingly induced following infection. The rise in GP73 expression coincided with the appearance of the adenovirus E1A and DBP proteins and preceded the expression of the fiber protein, a marker of the late phase of infection. Infection did not affect the expression of giantin, GPP130, or golgin-84, three integral Golgi membrane proteins with structural similarities to GP73. Mapping studies using a panel of mutant adenoviruses demonstrated that the E1A C-terminus, specifically its CtBP interaction domain (CID), is required for GP73 expression. Subsequently, Hep3B cells were transiently transfected with plasmids expressing wild-type or mutant E1A proteins. These studies confirmed that E1A induced GP73 expression via the CID. Our studies establish GP73 as a novel adenovirus-induced cellular protein whose expression is regulated through the CID of the E1A protein.     

Tumor necrosis factor-mediated interactions between inflammatory response and tumor vascular bed

Summary: Solid tumor therapy with chemotherapeutics greatly depends on the efficiency with which drugs are delivered to tumor cells. The typical characteristics of the tumor physiology promote but also appose accumulation of blood-borne agents. The leaky tumor vasculature allows easy passage of drugs. However, the disorganized vasculature causes heterogeneous blood flow, and together with the often-elevated interstitial fluid pressure, this state results in poor intratumoral drug levels and failure of treatment. Manipulation of the tumor vasculature could overcome these barriers and promote drug delivery. Targeting the vasculature has several advantages. The endothelial lining is readily accessible and the first to be encountered after systemic injection. Second, endothelial cells tend to be more stable than tumor cells and thus less likely to develop resistance to therapy. Third, targeting the tumor vasculature can have dual effects: (i) manipulation of the vasculature can enhance concomitant chemotherapy, and (ii) subsequent destruction of the vasculature can help to kill the tumor. In particular, tumor necrosis factor α is studied. Its action on solid tumors, both directly through tumor cell killing and destruction of the tumor vasculature and indirectly through manipulation of the tumor physiology, is complex. Understanding the mechanism of TNF and agents with comparable action on solid tumors is an important focus to further develop combination immunotherapy strategies.     

Chinese hamster cells deficient in ornithine decarboxylase activity: Reversion by gene amplification and by azacytidine treatment

A group of Chinese hamster ovary (CHO) cell mutants deficient in ornithine decarboxylase (ODC) activity are described and compared to the prototype mutant reported previously (21). Although all mutants belong to the same complementation group, they can be divided into two classes: those with some residual enzyme activity and those with no activity. All mutants are putrescine auxotrophs, but they differ in their ability to utilize the enzyme's substrate, ornithine, a property which correlates with the amount of residual enzyme activity. The mutants also differ in their frequency of reversion to prototrophy. The leaky mutants revert at a high rate by overproducing a partially defective enzyme by a gene amplification mechanism similar to that leading to the ornithine analog-resistant mutants which have elevated enzyme levels. Spontaneous reversion in the null mutants is rare. However, one null mutant, which was induced with ethyl methane sulfonate and which makes ODC mRNA but no active enzyme, is nevertheless revertible with 5-azacytidine. We conclude that CHO cells are at least diploid at the ODC locus, but that only one allele is active. Further studies suggest the possibility that ethyl methane sulfonate is not just a classical mutagen but may also induce gene inactivations that are revertible by 5-azacytidine.     

Modulation of cyclic nucleotide levels in peripheral nerve without effect on resting or compound action potentials.

1. Cyclic nucleotide levels and compound action potential magnitudes were measured in frog sciatic nerves following exposure to carbachol, isoprenaline and cyclic nucleotide related substances. 2. The resting cyclic AMP level was 2-4 p-mole/mg protein and the cyclic GMP level was 0-27 p-mole/mg protein in desheathed nerves. 3. Isoprenaline (100 micrometer) caused a twofold increase in cyclic AMP without affecting cyclic GMP levels. Carbachol (100 micrometer) caused a twofold increase in cyclic GMP without affecting cyclic AMP levels. 4. The phosphodiesterase inhibitor theophylline (5 mM) augmented both cyclic AMP and cyclic GMP. 5. The magnitude of the resting or compound action potential was not affected by isoprenaline, carbachol, or phosphodiesterase inhibitors. 6. The cyclic nucleotides and their butyryl derivatives did not affect the magnitude of the resting or compound action potential, either when applied alone or in the presence of a phosphodiesterase inhibitor. 7. In contrast to sympatic tissue we conclude that hormone mediated cyclic nucleotide metabolism in peripheral nerve is unrelated to control of axonal excitability.     

Comparison of Discrete Cardiovascular Fitness Groups on Plasma Catecholamine and Selected Behavioral Responses to Psychological Stress

Discrete cardiovascular fitness groups consisting of high-fit (n=10) and low-fit (n=9) men performed a well-learned vigilance task and their self-report, performance, and plasma catecholamine responses were compared. No significant differences were observed between the fitness groups on self-report or psychomotor performance responses to the vigilance task. However, the low-fit group took significantly longer than high-fit subjects to complete the first of three sets of anagrams administered immediately after the vigilance task. Plasma norepinephrine but not epinephrine response was greater in the low-fit group compared to their high-fit counterparts. The findings indicate that enhanced cardiovascular fitness may be characterized by an attenuated plasma norepinephrine response to a vigilance task with sustained cognitive performance subsequent to the task.     

Influence of lamella features of UHMWPE on its physical and uniaxial tensile properties. I. Effect of sterilization method in uncrosslinked and unaged materials

The changes in the tensile toughness (U), degree of crystallinity (%C), melting temperature (Tm), lamella thickness, and lamella alignment of uncrosslinked and unaged GUR 1150HP ultra-high-molecular-weight polyethylene specimens, following different sterilization treatments (none, gamma-irradiation in air, gamma-irradiation in a nitrogen-rich atmosphere, ethylene oxide gas, and gas plasma) were determined. For each of the properties - U, %C, and Tm - the only significant difference in the mean value was found between the set of specimens gamma-irradiated in air, on the one hand, and each of the other sets, on the other hand. It was found that lamella thickness showed little change between the groups of specimens but there was a significant difference in the lamella orientation between the set of specimens that had been gamma-irradiated in air, on the one hand, and each of the other sets, on the other. It thus appears that the changes seen in the physical and mechanical properties determined may be a reflection of the change in the polymer's lamella alignment.     

Predicting pregnancy and spermatogenesis by survival analysis during gonadotrophin treatment of gonadotrophin-deficient infertile men

BACKGROUND: Predictors of fertility or spermatogenesis during gonadotrophin therapy of gonadotrophin-deficient men remain poorly defined. METHODS AND RESULTS: In order to evaluate potential predictors, this study evaluated 29 consecutive gonadotrophin-deficient men all desiring paternity who received 43 courses of therapy in one centre between 1982 and 1998. The Kaplan-Meier survival analysis estimates of median (SE) time to a sperm concentration of >0, >5 and >20 x 10(6)/ml were 5.5 (1.1), 12.4 (2.3) and 29.1 (1.9) months respectively. Conception occurred in 22/43 cycles (with eight men achieving two pregnancies) with a median (SE) Kaplan-Meier estimate of 20.5 (4.7) months. The median sperm concentration at conception was 5.0 (SE 2.0; range 0.0-59.5) x 10(6)/ml. Multivariate correlated Cox proportional hazards models predicting these same sperm thresholds and conception were developed by forward stepwise variable selection with verification of the model by backward stepping. Larger testicular volume, prior gonadotrophin therapy, completion of puberty, older age, the absence of adverse fertility factors and the absence of multiple pituitary hormone deficiency predicted a favourable response. Multivariate modelling suggests that the two most important predictors of sperm output are testicular volume and pubertal status. The most important potentially modifiable predictor was prior gonadotrophin therapy. The efficacy of recombinant and urinary FSH were similar. Prior androgen therapy and partner's age did not appear to be significant. CONCLUSIONS: Since prolonged treatment may be required to induce spermatogenesis, attention to these predictors may allow appropriate early use of advanced reproductive technologies.     

Cytotoxicity of Hemolytic, Cytotoxic Necrotizing Factor 1-Positive and -Negative Escherichia coli to Human T24 Bladder Cells

Approximately one-half of Escherichia coli isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates. To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1⁺, and CNF1⁻ cystitis strains toward human T24 bladder epithelial cells in vitro. A total of 29 isolates from two collections of cystitis-associated E. coli were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain. Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1⁺ isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1⁻ strains in that collection (P = 0.009), but this pattern was not observed among isolates from the second collection (18 strains). To directly evaluate the role of CNF1 in cytotoxicity of hemolytic E. coli without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1. Compared to the CNF1⁺ parental isolates, no change in cytotoxicity was detected in these cnf1 mutants. Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic E. coli to damage human bladder cell monolayers in vitro.