In the present study, we have examined the differences among Corynebacterium parvum-activated, thioglycollate-elicited, and resident macrophages from (C57BL/6 X C3H)F1 mice utilizing two microcytotoxicity assays. Macrophage and Naegleria fowleri were interchangeably employed as effector and target cells. To assess the ability of the macrophage to lyse N. fowleri, the amoebae were labeled with 5 μCi of 3H-uridine per 5 x 10^5 cells for 18 h prior to co-culturing with adherent macrophage monolayers. Although the macrophage-induced lysis was density and time dependent, C. parvum macrophages consistently demonstrated an increased cytotoxic response to N. fowleri after 24 h co-incubation at a 10:1 effector-to-target ratio. In contrast, thioglycollate and resident macrophages were unable to elicit a cytotoxic response to N. fowleri. To ascertain the ability of N. fowleri to destroy the three macrophage populations, a 51cr post-labeling technique was employed. After 24 h of co-incubation, uptake of 51cr into remaining C. parvum-activated and thioglycollate-elicited macrophages exceeded that for resident macrophages. Parallel electron microscopic studies confirmed simultaneous destruction of both amoebae and macrophages. Concurrent studies utilizing cell-free lysates of N. fowleri displayed the same divergent spectrum of macrophage destruction for the different macrophage populations as was seen with N. fowleri trophozoites. Catalase is an enzyme which breaks down hydrogen peroxide, a chemical released from activated macrophages, which could contribute to macrophage-induced cytotoxicity. Leupeptin is an inhibitor of some serine and cysteine proteases. Activated macrophages also secrete proteases which may contribute to macrophage-induced cytotoxicity. Catalase, leupeptin, or hydrogen peroxide were added to macrophage cultures or N. fowleri lysate prior to coincubation or during co-incubation to determine possible roles of hydrogen peroxide and proteases in macrophage susceptibility to N. fowleri lytic substances. Exogenously added hydrogen peroxide had no effect on resident macrophage survival. Catalase and leupeptin produced effects unique for the macrophage cell type, with survival closely correlating to the types and quantities of cytotoxic substances associated with the macrophage. Results suggest that macrophage susceptibility to N. fowleri lysate may be due to the absence or presence of two or more macrophage components.

Expression levels of GABAA receptor subunits and activity of the Ca++ /calmodulin-dependent protein kinase II (CaM kinase II) enzyme system were evaluated in an in vitro hippocampal neuronal culture model of epilepsy. Treatment of hippocampal neuronal cultures with Mg++-free media for 3 hours results in the induction of an enduring "epileptic" state as evidenced by the expression of spontaneous recurrent seizure (SRS) discharge. The induction of the SRS activity was shown to be a N-methyl-D-aspartate (NMDA) receptor, Ca++-dependent mechanism (Sombati and DeLorenzo, J Neurophys., 73 (4), 1995). Significant and long-lasting decreases in mRNA expression for the GABAA α2 and α5 receptor subunits were observed in association with the induction of SRSs in this model, while levels for α1, β2 and γ2 subunits showed no significant change. Irreversible [3H]-flunitrazepam saturation binding analysis in membrane preparations demonstrated a significant decrease in specific binding in association with the SRS activity observed in this model. No changes in GABAA P subunit immunoreactivity were detected. Selective suppression of the GABAA α2 subunit protein levels in hippocampal neuronal cultures using antisense oligonucleotide technology caused a significant decrease in the amplitude of spontaneous inhibitory postsynaptic currents (sIPSC). CaM kinase II is highly enriched in the brain and mediates many processes essential to neuronal function and viability. Induction of SRSs in hippocampal cultures were associated with a long-lasting and significant decrease in activity of CaM kinase II. Addition of 2-amino-5-phosphovaleric acid to the low Mg++ treatment solution prevented the decrease in CaM kinase II activity. Suppression of CaM kinase II activity in hippocampal cultures by treatment with either an antisense oligonucleotide specific for a CaM kinase II or KN93 (selective CaM kinase II inhibitor) resulted in significant reductions in IPSC amplitude. This data suggests that CaM kinase II can act to regulate GABAergic inhibitory function in hippocampal cultures. The findings of this study demonstrate long-lasting decreases in GABAA receptor expression and activity of CaM kinase II, which may contribute to the induction of the "epileptic" state of this model.

An HPLC prodecure was developed for the simultaneous measurement of imipramine (IMP), desipramine (DMI), desmethyl desipramine (DMD), 2-hydroxyimipramine (2HI) and 2-hydroxydesipramine (2HD) in human, rat and rabbit blood and brain specimens. The HPLC results were validated by comparison to an established GC-MS method. DMD is a minor metabolite of IMP and DMD in humans. 2HI is a minor metabolite of IMP in most humans. 2HD reaches appreciable concentrations in blood in humans treated with DMI and IMP. Plasma and RBC concentrations of IMP and DMI were compared to improvement in depression in patients treated with IMP. No good correlation was found. Plasma and RBC concentrations of DMI were compared to improvement in depression in patients treated with DMI. Plasma concentrations (r = -0.750, p < .01) and RBC concentrations (r = -0.693, p < .01) correlated with improvement in depression. As plasma DMI increased past 400 ng/ml, the degree of improvement declined. In autopsy specimens from an IMP overdose, IMP, DMI, DMD, 2HI and 2HD were all measured in whole blood and brain. In autopsy specimens from b/0 DMI overdoses, DMI, DMD and 2HD were all measured in whole blood and brain. The brain to blood ratios in all cases for IMP and DMI were significantly higher than the brain to blood ratio of 2HD, the most polar metabolite. This finding indicates that 2HD has restricted entry into the brain compared to IMP and DMI. The distribution of 2HD between plasma and brain was studied in rats. The mean brain to plasma ratio for DMI was 5.87 and for 2HD it was 0.067. Therefore, in rats 2HD has limited access into brain from blood compared to DMI. In rat studies, both plasma and RBC concentrations of DMI correlated well with brain DMI concentrations. In humans and rats, the plasma to RBC ratios for IMP and DMI are significantly different. IMP has greater affinity for plasma than RBCs. DMI is the reverse, having greater affinity for RBCs than plasma. As indicated, for DMI treated patients, plasma DMI concentrations correlated slightly better than RBC DMI concentrations to patient improvement in depression.

Arterial disease is virtually a universal entity in man. Some cultures, such as the African Bantus have very little in the way of vascular lesions, while in the United States and other industrialized nations atherosclerosis affects a large segment of the population. With this in mind it is not hard to understand why ischemia, or arteriosclerotic heart disease, is second only to cardiovascular diseases in general as the major cause of death in the United States (4, 42). Ruffer (112) noted the presence of arterial lesions in Egyptian mummies dating back 3,400 years. The presence of atherosclerotic lesions has also been reported in a 2100 year old Chinese mummy (36). Though atherosclerosis is an ancient disease it is seen more and more often in our culture in vascular disease. The early sixteenth and seventeenth century anatomists were the first to make note of arterial lesions and guess at their origins. Over the centuries came theories and definitions that are at the least confusing. Virchow in 1856 (6, 7, 45 ) was the first to propose the theory of mechanical damage to the intima of the artery to account for the entry of blood plasma into the arterial wall. The lesions were attributed to chronic irritation. Marchand in 1904 coined the term atherosclerosis. At the time the dominant concept was one of arterial lesions forming after there was an increase in sub-intimal connective tissue due to irritative or mechanical forces. Today much more is understood about the processes of arteriosclerosis, but definitions still vary among authors. Arteriosclerosis is the broad generic term which literally means "hardening of the arteries" and involves the processes of thickening and loss of elasticity to arterial walls (42, 108, 109). Three distinct morphological entities are grouped under this broad heading; (1) medial sclerosis, (2) arteriolosclerosis and (3) atherosclerosis. The medial or Monckeberg’s sclerosis is characterized by calcifications within the media of medium to small arteries, particularly the femoral, tibial, radial and ulnar arteries. Arteriolosclerosis is particularly common in hypertension and affects the small arteries and arterioles. It is distinguished by hyaline proliferation and fibrous and elastic hyperplasia of the media and the intima. Atherosclerosis is the most commonly seen form of the arteriosclerotic diseases and when it occurs it has the most serious clinical consequenses. The aorta and other large elastic arteries of the extremities are the primary locations for atherosclerosis. The disease is characterized by patchy, nodular lesions due to a thickening of the intima and a degenerative process in which lipid deposition occurs. The media may also become involved. The earliest descernible lesion is termed a fatty streak. It presumably occurs over an area of stress to the vessels. Lipid is deposited in the site and soon macrophages and myocytes become filled with the fat droplets and form "foam cells". The intima is thickened and the elastic lamina is disrupted by extracellular fat. As the pathogenesis continues the arterial tissue attempts to repair itself and a fibrous capsule is layed down over the lesion thickening the intima even more. The term fibrous or pearly plaque now differentiates the lesion. The cells in the center of this lesion die due to a lack of oxygen and a necrotic mass, termed "gruel" is formed. From the vasa vasorum large capillaries or "sinusoids" invade the gruel and at the same time calcium is being deposited and the lesion can become calcified or "hardened". Up to this point the body has been doing though the usual healing and repair processes, but now destructive changes can occur. The cracking or breaking away of a plaque can cause hemorrhage or ulceration. These degenerative changes can be a grave threat when aneurysm or thrombus formation occurs. It appears that this sequence of lesion formation must occur in waves throughout a lifetime since lesions of various size and age can be found side by side in the aorta. In general however, there is agreement that in North America at least the fatty streaks appear during the first decade of life while pearly plaques are forming in the second and third decade. The complications and their consequences occur from the fourth decade on (16, 21, 72).

Control of hematopoiesis is a complex set of events that is currently being dissected at the molecular level. To determine factors that may be crucial for commitment to terminal differentiation of myelomonocytic cells, a mutant of the HL-60 cell line was characterized at the cellular and molecular level. This clone, termed DMSOr, was shown to differentiate in a similar fashion as parental HL-60 in response to 1.3% DMSO at the morphologic and functional level. The anti-proliferative aspects of differentiation were also present in DMSOr as evidenced by decreased 3H-thymidine incorporation and an increased percentage of cells in the G0/G1 phase of the cell cycle. All of these phenotypic changes induced in DMSOr would revert if the DMSO was removed at any point during the differentiation process, thus DMSOr, despite its ability to functional differentiate, could not commit to terminal differentiation. Associated with the altered phenotype of DMSOr was the altered expression of the proto-oncogene c-myb. Expression of cmyb remained detectable at 144 hrs of DMSO treatment in DMSOr but not HL-60. Similar findings were shown for the cell cycle other related genes. This altered gene expression did not extend to the c-myb related gene B-myb. The possibility that altered transcriptional regulation of c-myb was eliminated by nuclear run-on analysis and by the fact that a splice variant of c-myb with an altered 3' untranslated region showed no altered regulation. Thus the genetic defect in DMSOr may be in a global control factor for cell cycle related genes such as c-myb. This factor may regulate these genes at the post-transcriptional level. To determine the mechanisms of regulation of c-myb during hematopoietic cell differentiation, transcriptional and posttranscriptional studies of c-myb following treatment of HL-60 cells with various differentiation inducers were undertaken. Retinoic acid and vitamin D3 regulated c-myb at the transcriptional level via an attenuator, while DMSO and phorbol dibutyrate activated multiple mechanisms of regulation. These included attenuation and a post-transcriptional regulation that was dependent on continuous transcription, but not translation in the case of DMSO. Phorbol ester regulation of c-myb occurred at the level of an attenuator and possibly a promoter at the transcriptional level. In addition there was post-transcriptional control of c-myb by phorbol dibutyrate that differed from the regulation by DMSO through the lack of transcriptional dependency. Thus c-myb is regulated at the transcriptional and the post-transcriptional level in an agent specific fashion during HL-60 differentiation. A 2.4 kb message is present in the Northern blots probed for c-myb expression. This lower molecular weight message is regulated in an abberrant fashion compared to normal message during HL-60 differentiation. Probing of blots with different regions of a full length c-myb eDNA and primer extension analysis suggest that the 2.4 kb message may start in the exon of the c-myb locus.

Phencyclidine (PCP) belongs to a class of drugs with a unique and characteristic spectra of pharmacological activity. PCP has recently become a major drug of abuse. Structural analogues of PCP have also been reported in street use. The following experiments explore the behavioral pharmacology of PCP and several compounds with PCP-like activity. In Experiment I the effects of PCP were compared to three structural analogues in rhesus monkeys trained to lever press on a fixed-interval 5 min schedule of food presentation. Dose-response curves and potency estimates were determined for PCP, N-ethyl-1-phenylcyclohexylamine (PCE), 1-(1-(2-thienyl) cyclohexyl) piperidine (TCP) and ketamine.The effects of all four drugs on response rates were dependent on the baseline rate of responding. High doses of all four drugs decreased overall response rates. Onset and duration of drug effects were also determined and compared. In Experiments II through VI the drug discrimination paradigm was used. Animals were trained to discriminate PCP (rats, 3.0 mg/kg i.p.; monkeys, 0.16 mg/kg i.m.) from saline in a two-lever drug discrimination task on a fixed-ratio 32 (FR 32) schedule of food presentation. After reliable discrimination control of lever choice was established, generalization tests were conducted every third day. Test periods consisted of a 2-min period during which responding on either lever was reinforced (rats) or responding was recorded but not reinforced (monkeys). The PCP dose-response determination preceded generalization testing with other compounds. Dose-effect curves for each drug for percent drug-lever appropriate responding and for suppression of operant responding during sessions were analyzed by linear regression. In Experiment II the discriminative stimulus properties of PCP and five other arylcycloalkylamines were investigated in squirrel monkeys .Generalization testing was conducted with PCP, PCE, TCP, 1-(1-phenylcyclohexyl) morpholine (PCM), 1-(1-phenylcyclohexyl) pyrroldine (PHP), and ketamine. All drugs produced dose-dependent PCP-appropriate responding. The dose necessary to suppress operant responding to fifty percent of vehicle rates was 3 to 8 times larger than the ED50 for drug-lever appropriate responding. Experiments III, IV and VI were designed to explore similarities between the discriminative stimulus properties of PCP and a series of structurally dissimilar compounds. In Experiment III the discriminative stimulus properties of dexoxadrol and etoxadrol, both 2-(2,2-substituted 1,3 dioxolan-4-yl) piperdines, were studied in squirrel monkeys. Both compounds generalized to PCP in a dose-dependent manner. The dose necessary to suppress operant responding to fifty percent of vehicle rates was 3 to 8 times larger than the ED5O dose · for drug-lever appropriate responding. In Experiment IV the discriminative stimulus properties of stereoisomers of N-allylnormetazocine, a benzomorphan opioid with psychotomimetic properties, were investigated in squirrel monkeys and rats. In both species, the (+) isomer and the racemic mixture produced dose dependent PCP-appropriate responding. The (-) isomer did not produce PCP-appropriate responding, however it was more potent than the (+) isomer in overall response rate suppression. High doses of naloxone (rats, 30 mg/kg;monkeys, 1.0 and 3.0 mg/kg) did not block the drug lever appropriate responding or response rate suppression produced by either isomer of N -allynormetazocine. In Experiment V the discriminative stimulus properties of ketamine stereoisomers were investigated in rats. Both isomers and the racemic form of ketamine produced drug-lever appropriate responding and decreased response rate in a dose-dependent manner. There was no significant potency difference between (±)-ketamine and either of the isomers for either of these measures. The stereoselectivity of the discriminative stimulus properties of cyclazocine, another opioid of the benzomorphan series, in squirrel monkeys was explored in Experiment VI. The (+) isomer produced dose dependent PCP-appropriate responding . The (-) isomer and the racemic mixture did not produce PCP-appropriate responding at any of the doses tested. The (-) isomer was 300 times more potent than the (+) isomer in overall response rate suppression. The work presented in this thesis suggests that PCP belongs to a unique class of drugs which can have a wide variety of chemical structures. The overalp in the behavioral pharmacology of PCP and the psychotomimetic opioids of the benzomorphan series is probably due to the activity of the (+) isomers of these compounds.

In order to prepare more effective inhibitors of ribonucleotide reductase a series of 4-substituted and 4,5-disubstituted catechols were synthesized and tested. The derivatives synthesized were also examined for their antitumor activity against L1210 leukemia in mice. A free radical scavenging assay was performed to establish what electronic parameters may govern in vitro and/or in vivo activity. The molecular features of the most potent compounds were compared to the features of the natural substrate of reduction, CDP, UDP, GDP and ADP. The results obtained from the free radical assay did not show any correlation to the results observed either in vitro or in vivo. Enhanced activity, both in vitro andin vivo, was shown when the amide function of 3,4-dihydroxybenzamide was substituted with a hydroxyethyl or a dimethylaminoethyl side chain. Reversal of the amide bond, ex. N-(3,4-dihydroxyphenyl)acetamide, resulted in a log unit increase as an inhibitor of ribonucleotide reductase. Antitumor activity was also substantially increased. The most potent compounds found in this study appear to physically resemble the molecular features found in the natural substrates.

Twenty-six undergraduate medical technology students at Virginia Commonwealth University were provided with a Hemestasis Study Guide to use during this unit and in preparing for an exam in this area. The study guide contained fifty objective-related questions in multiple-choice format. Answers were printed in latent image and included positive reinforcement for correct selections and remediation for incorrect ones. Thus the student received immediate feedback concerning his response to each question. An examination grade continuum was plotted for this class utilizing all exam grades in the Hematology course. A similar continuum was plotted for each of the three previous classes. (These classes served as control groups.) A positive peak between the hemostasis exam grade and the grade of the prior exam was noted for the experimental class. The three previous classes also showed the same positive peak. In order to determine whether the amount of gain obtained by the experimental class was significant, an unpaired t-test was used to compare this gain with each of the three previous classes. The gain of each of the control classes was also compared with the other control classes using the same test. The gain of the experimental group was significant when compared with the gains of eaoh of the previous three control classes at the 99% confidence interval. Ho significance was observed between the control classes. These results suggest that learning was enhanced in the experimental group when compared with the control groups. It is probable that this enhanced learning was due to utilization of the study guide and/or immediate corrective feedback.

In metabolic acidosis, the early increase in PEPCK mRNA and enzyme protein content contributes to the accelerated rates of ammonium and glucose formation. In situ hybridization demonstrated that expression of PEPCK was confined to medullary rays of rat kidney cortex in controls and spread throughout the cortex 10 hours following NH4Cl feeding (Am.J. Physiol., 267: F400, 1994). To identify the specific nephron segments expressing PEPCK in control and acidotic conditions, the mRNA for PEPCK along the nephron of the rat kidney was localized using the technique of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected S1, S2 and S3 segments of the rat proximal tubule. Two-millimeter segments of tubule were permeabilized, the mRNA reverse transcribed using oligo-dT as a downstream primer and the eDNA product amplified by PCR (35 cycles). Primers specific for β-actin were used to confirm transfer of tubule, and only tubules positive for β-actin were amplified with primers specific for PEPCK. Both primers were designed to span at least one intron to avoid amplification of genomic DNA. The PCR products were detected using agarose gel electrophoresis and ethidium bromide staining. Verification of PCR product was performed by restriction enzyme digestion. Under control conditions, the number of tubules expressing PEPCK mRNA as detected by RT-PCR was greatest in the S3 segment, moderate in the S2 segment, and least in the S1 segment of the proximal tubule. Ten hours after gavage feeding of 20- mmol/kg bodyweight NH4Cl, strong signals for PEPCK were detected in all three proximal tubule segments. These data demonstrate the ability of the rat kidney cortex to modulate the expression of PEPCK mRNA along the proximal tubule under physiological conditions, and to increase expression of PEPCK mRNA during metabolic acidosis by the recruitment of additional cells in the proximal nephron.

The present report describes the characteristics and regulatory properties of phosphatidylinositol (Ptdlns) 4-kinase activity in rat exocrine pancreas. The membrane associated Ptdlns 4-kinase displayed a broad pH profile with optimal activity at neutral to alkaline pH. Carbachol (CCh) elicits a concentration- and time-dependent increase in Ptdlns 4-kinase activity in homogenates derived from agonist-stimulated acini. This effect was blocked by N-methylscopolamine and mimicked by muscarine. The enzyme had an apparent Km for Ptdlns and ATP of 4 and 60 uM, respectively. CCh caused no discernible change in the Km for either Ptdlns or ATP, but did produce a modest increase in the Vmax. The cell permeable diacylglycerol analogues dioctanoylglycerol (DiC8) and oleoyl acetylglycerol (OAG) also produced a concentration-dependent increase in Ptdins 4-kinase activity which was blocked by the protein kinase c inhibitor, staurosporine. Cell permeable monobutyryl cAMP caused an increase in PtdIns 4-kinase activity when added to intact acini. This agent caused a significant decrease in the apparent Km for ATP but had no effect on PtdIns utilization or the Vmax of the reaction. Moreover, pretreatment of the acinar homogenate with the catalytic subunit of protein kinase A (PKA) produced a concentration-dependent increase in enzyme activity suggesting that phosphorylation may be involved in the regulation of Ptdins 4-kinase. Epidermal growth factor (EGF) elicited a concentration-dependent increase in PtdIns 4-kinase activity in homogenates derived from agonist-stimulated pancreatic acini. The combination of CCh and EGF produced a response which was not synergistic or additive. EGF, unlike CCh, failed to cause [32^P]Ptdins(4,5)P2 breakdown, suggesting different mechanisms for the increase in Ptdins 4-kinase activity induced by EGF and CCh. Furthermore, exposure of pancreatic acinar cells to EGF failed to elevate cyclic AMP levels, suggesting that a third pathway exists for the regulation of Ptdins 4-kinase activity in the exocrine pancreas. We conclude that PtdIns 4-kinase represents a key component of the signaling pathways utilized by various receptor agonists and that phosphoinositide synthesis is under direct and tight regulation by PKC- and PKA-dependent pathways.