Search

Identifying neurobiological mechanisms of aging-related parkinsonism, and lifestyle interventions that mitigate them, remain critical knowledge gaps. No aging study, from rodent to human, has reported loss of any dopamine (DA) signaling marker near the magnitude associated with onset of parkinsonian signs in Parkinson’s disease (PD). However, in substantia nigra (SN), similar loss of DA signaling markers in PD or aging coincide with parkinsonian signs. Alleviation of these parkinsonian signs may be possible by interventions such as calorie restriction (CR), which augment DA signaling markers like tyrosine hydroxylase (TH) expression in the SN, but not striatum. Here, we interrogated respective contributions of nigral and striatal DA mechanisms to aging-related parkinsonian signs in aging (18 months old) rats in two studies: by the imposition of CR for 6 months, and inhibition of DA uptake within the SN or striatum by cannula-directed infusion of nomifensine. Parkinsonian signs were mitigated within 12 weeks after CR and maintained until 24 months old, commensurate with increased D1receptor expression in the SN alone, and increased GDNF family receptor, GFR-α1, in the striatum, suggesting increased GDNF signaling. Nomifensine infusion into the SN or striatum selectively increased extracellular DA. However, only nigral infusion increased locomotor activity. These results indicate mechanisms that increase components of DA signaling in the SN alone mitigate parkinsonian signs in aging, and are modifiable by interventions, like CR, to offset parkinsonian signs, even at advanced age. Moreover, these results give evidence that changes in nigral DA signaling may modulate some parameters of locomotor activity autonomously from striatal DA signaling.

Reaction of RhCl(CO)22 with a phosphine/thioether/borane ligand (TXPB) gave Rh(-Cl)(CO)(TXPB) (1), a rare example of a complex containing a M-Cl-BR3bridging interaction. Reaction of 1with KCpFe(CO)2 gave (TXPB)Rh(-CO)2Fe(CO)Cp (2), which contains an unprecedented 3-interaction between rhodium and the B-Cipso-Corthounit of a triarylborane. DFT calculations suggest a bonding description intermediate between that expected for an isolated borane/alkene complex and a fully delocalized allyl-like complex.

Expression of the human DNA topoisomerase IIα (topo IIα) gene is positively regulated by the binding of the nuclear factor Y (NF-Y) transcription factor to four of five inverted CCAAT boxes (ICBs) located in its promoter. We have demonstrated previously that expression of the p53 tumor suppressor inhibits human topo IIα promoter activity in murine (10)1 cells. In this report, we demonstrate that the inhibition of topo IIα gene expression by wild-type p53 correlates with the decreased binding of the transcription factor NF-Y to the first four ICBs of the topo IIα promoter. The expression of mutant p53 does not affect the binding of NF-Y. In NIH3T3 cells, we show that topo II-targeted drugs inhibit the binding of NF-Y to ICB sites in the topo IIα promoter. This effect is seen not only with drugs that result in DNA strand breaks but also with drugs that inhibit the catalytic activity of topo II, and even with the mitotic spindle inhibitor, vinblastine. Further experiments with p53-null (10)1 cells treated with these same drugs also demonstrate decreased NF-Y binding to the topo IIα ICBs. The data presented points to the existence of both p53-dependent and -independent mechanisms for regulating NF-Y binding to ICBs in the topo IIα promoter and thus the modulation of topo IIα gene expression.

Levels of endothelin-1 (ET-1) are elevated in many disease states, although its total body kinetics of elimination are poorly understood. Therefore, it remains uncertain whether the presence of elevated levels of ET-1 in the setting of disease are secondary to changes in production or clearance or some combination thereof. Using a 125I-labeled ET-1 infusion technique, the volume of distribution and kinetics of clearance of endothelin were described in five normal volunteers. Heart rate, blood pressure, right atrial pressure, and arterial blood samples for the counting of 125I and the measurement of ET-1 were obtained at multiple time points before and up to 45 h after the start of the infusion. The radiotracer infusion had no effect on heart rate, blood pressure, right atrial pressure, or endogenous ET-1 levels. ET-1 clearance was best described by a three-compartment model, which revealed that ET-1 has a much longer terminal half-life and volume of distribution than was previously reported. This suggests extensive uptake of ET-1 in various organ systems and slow clearance. These new findings have important implications for the understanding of the pathophysiology of ET-1 in disease states as well as for the understanding and development of ET-1 receptor blockers and endothelin-converting enzyme inhibitors.

Reversible oxidation potentials, rate constants, and activation parameters are reported for the formation and decomposition reactions of all isomers of (methyl-substituted benzene)tricarbonylchromium cation radicals. The reversible potentials were observed to depend both on the number and the positions of the methyl substituents. It was found that the effects on the reversible potentials of adding single methyl substituents to similar structures were additive. The activation energies and entropies for the decomposition reactions were found to be grouped into four different categories depending upon the numbers of adjacent unsubstituted positions on the methylarene ligands. Within each group the deviations from the averages were less than experimental error. The differences in activation parameters are explained in terms of how effective bonding is between the chromium atom and the arene ligands in the cation radicals.

Orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC), which catalyze the last two steps in de novo UMP biosynthesis, are two distinct monofunctional proteins in bacteria and lower eukaryotes. In mammals, OPRT and ODC activities are contained in a single bifunctional protein labeled UMP synthase. The human UMP synthase cDNA was separated into the predicted OPRT and ODC domains using polymerase chain reaction techniques and the domains inserted into pUC19 expression vectors. Following transformation into OPRT- and ODC-deficient E. coli, the strains were able to grow on minimal media without uridine. The ODC-transformed bacteria expressed up to 24 times the level of activity found in a wild-type E. coli line. The OPRT-transformed E. coli contained only 4–9% of wild-type activity. Western blot analysis with antiserum to human UMP synthase demonstrates that OPRT and ODC domains are being produced in the deficient cells by the respective vectors. The level of the domain protein approximates the level of enzyme activity. The complementation of the OPRT and ODC activities in the transformed deficient E. coli strains demonstrates that human UMP synthase can be separated into active monofunctional domains that will function in the bacterial cell environment.

It has been reported that nitroglycerin (GTN) tolerance can be prevented by the concurrent administration of hydralazine. Although the mechanism of this effect remains unknown, it is possible that hydralazine modifies counter-regulatory responses to nitrate administration. To address this question, we examined the impact of hydralazine therapy on the development of tolerance during sustained therapy with GTN. Twenty normal volunteers and 18 patients with chronic heart failure (mean ejection fraction 30 +/- 2%) were treated for 1 week with hydralazine or placebo in a randomized double-blind fashion. Hydralazine therapy (or placebo) was continued, and subjects then received continuous transdermal GTN for 5 to 7 days. On the first and last day of transdermal GTN therapy, standing HR, systolic blood pressure and hematocrit responses were assessed. HR and blood pressure responses to sublingual GTN (0.6 mg) were also evaluated before and during sustained transdermal GTN therapy. Significant loss of the hemodynamic effects of transdermal GTN occurred during sustained therapy in both the normal volunteer and heart failure groups. Hydralazine had no effect on the development of tolerance to the hemodynamic effect of GTN in either group. In both, transdermal GTN therapy was associated with a significant fall in hematocrit that persisted for the entire treatment period. Hydralazine had no effect on this response. These data suggest that hydralazine therapy does not prevent loss of systemic arterial effects or prevent plasma volume expansion during sustained transdermal GTN therapy.

DNA topoisomerase II (topo II) is an essential nuclear enzyme involved in major cellular functions such as DNA replication, transcription, recombination, and mitosis. While an elevated level of topo II alpha is associated with cell proliferation, wild-type (wt) p53 inhibits the expression of various growth-stimulatory genes. To determine if p53 downregulates topo II alpha gene expression, a murine cell line, (10)1val, that expresses a temperature-sensitive p53 was utilized. The (10)1val cells had significantly lower levels of topo II alpha mRNA and protein following incubation for 24 h at 32°C (p53 with wt conformation) than at 39°C (p53 with mutant conformation). The effect of p53 on the human topo II alpha gene promoter was determined by using luciferase reporter plasmids containing varying lengths of the topo II alpha promoter transiently cotransfected into p53-deficient (10)1 cells together with wt or mutant p53 expression plasmids. Transcription from the full-length (bp −557 to +90) topo II alpha promoter was decreased 15-fold by wt p53 in a concentration-dependent manner, whereas mutant p53 exerted much weaker inhibition. Consecutive deletion of the five inverted CCAAT elements (ICEs) from the topo II alpha promoter reduced both the basal promoter activity and wt p53-induced suppression. Transcription of the minimal promoter (−32 to +90), which contains no ICE, was slightly stimulated by wt or mutant p53 expression. When point mutations were introduced into the most proximal ICE (−68), the inhibitory effect of wt p53 was alleviated and stimulation of topo II alpha expression resulted. Our study suggests that wt p53 functions as a transcriptional repressor of topo II alpha gene expression, possibly through a functional interaction with specific ICEs. Inactivation of wt p53 may reduce normal regulatory suppression of topo II alpha and contribute to abortive cell cycle checkpoints, accelerated cell proliferation, and alterations in genomic stability associated with neoplasia.

Chinese hamster lung cells selected for resistance to pyrazofurin and 6-azauridine contain amplified UMP synthase genes. With selection in 5-fluorouracil, cells that have lost the amplified gene copies can be isolated. Reselection of deamplified cells in pyrazofurin and 6-azauridine results in reamplification of the UMP synthase genes. We have used chromosomal banding and in situ hybridization techniques to characterize this cyclic process of amplification and deamplification. Homogeneously staining regions (HSRs) were observed in cells containing amplified copies of the UMP synthase gene but not in cells in which the amplified UMP synthase genes had been lost. After reselection in pyrazofurin and 6-azauridine, abnormally banded regions (ABRs) were observed. Both HSRs and ABRs were located at a single site on the distal regions of a small acrocentric autosome, and both were shown to contain the amplified genes. The majority of 5-fluorouracil-selected cells showed residual marker acrocentric chromosomes of various sizes, suggesting excision of portions of the HSR or ABR as the mechanism of deamplification. The acrocentrics carrying the amplified genes resulted from rearrangements involving chromosome 4, site of the endogenous gene. This reversible selection system provides a unique model for investigating gene amplification and deamplification in association with chromosomal rearrangements and the relationship of G-banding to underlying DNA structure.

The bifunctional enzyme UMP synthase provides a unique reversible selection system whereby cells that have amplified the UMP synthase gene can be isolated from a wild-type population and cells that have deleted the extra genes can be selected from a population with amplified copies of the gene. UMP synthase catalyzes the conversion of orotic acid to orotidine 5'-monophosphate (OMP) and then OMP to UMP. In the amplification step, Chinese hamster lung cells are selected for resistance to pyrazofurin and 6-azauridine, two inhibitors of the orotidine 5'-decarboxylase activity that converts OMP to UMP. The resistant cells have increased levels of both activities of UMP synthase as a result of a stable amplification of the UMP synthase gene. The deamplification step depends on 5-fluorouracil (5FU), which is converted to its monophosphate form by the orotate phosphoribosyltransferase activity of UMP synthase. Thus cells with increases in this activity are more sensitive to 5FU cytotoxicity, permitting single-step selection of revertants that have lost their amplified UMP synthase genes. These 5FU-selected cells are similar to the parental cell line in their level of UMP synthase activity and number of UMP synthase gene copies. Reselection in increasing concentrations of pyrazofurin and 6-azauridine allows one to isolate cells that have reamplified the UMP synthase gene. The ability to cycle cells of a single lineage through states of amplification and deamplification will facilitate study of the gene amplification process and the factors that influence the composition and stability of amplified regions.

Three different phenotypes have been characterized in HeLa cells that have been selected for resistance to pyrazofurin, a potent inhibitor of the de novo pyrimidine biosynthetic enzyme UMP synthase. All of the resistant cell lines had a coordinate increase in UMP synthase activity, UMP synthase-specific mRNA, and UMP synthase gene sequences. In one of the resistant cell lines, the amplification of the UMP synthase gene is associated with a stable phenotype. There is no decrease in UMP synthase gene copy number or UMP synthase activity when these cells are grown for over six months in the absence of pyrazofurin. Another resistant cell line that has a higher level of gene amplification when grown in the presence of pyrazofurin loses its elevated UMP synthase activity and amplified DNA sequences with growth in the absence of the drug. A third cell line that possessed a moderate level of UMP synthase gene amplification is tenfold more resistant to pyrazofurin than the cell line with the highest level of amplification. The extraordinary level of resistance is due to a decreased level of activity for the enzyme adenosine kinase that is required for the conversion of pyrazofurin to its inhibitory monophosphate form.

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo IIa gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected. We propose that MDR associated with alterations in topo II may have clinical consequences, and our current efforts involve exploiting these biochemical and molecular observations in the development of probes that may be useful to identify such drug resistant cells in the tumors of patients.

A 94-year-old female presented to the emergency department with acute expiratory stridor. In the absence of an otorhinolaryngologist, an urgent laryngoscopy was performed using a flexible bronchoscope by an anaesthesiologist in the emergency department leading to a change in management. Subsequent radiographs confirmed severe tracheal compression from megaoesophagus secondary to achalasia as the cause of acute airway obstruction. Use of flexible bronchoscope as a diagnostic tool by an anaesthesiologist to evaluate a patient presenting with signs of acute airway obstruction may lead to a safer and more careful airway management planning. Suggestions are also made regarding establishment of emergency surgical airways when conventional approaches fail.

An asymmetric route from the epimeric -hydroxy esters 4and 5to the densely functionalized ()-10and (−)-10, respectively, is described. Either cyclobutanol can be made available as the predominant product. The levorotatory antipode has been transformed into the advanced intermediate 21bearing side chains destined to become incorporated into the cyclononene ring of the title compound (1).

Statistical Layer Shift, α-Chitin The intensities scattered by antiparallel/parallel layers were calculated. Difference synthesis maps allowing for statistical layer shifts were computed for a-chitin. The results support the view that there may be antiparallel sequence with each layer displaced at random by +e or -e from a halving position in the ϰ-direction. This is supported by greater breadth of certain reflection of the type lkl compared to 0kl on the X-ray diffraction photograph. This gives strong evidence for some imperfection in the ϰ-direction and the proposed indeterminacy + e is one possible way in which this might arise.

Research indicates that contingency management (CM) has potential to improve a number of outcomes (e.g. substance use, treatment attendance, quality of life) among individuals with substance use and cooccurring disorders. However, multiple factors must be considered on a case-by-case basis in order to promote optimal treatment effects. The present study describes an individualized CM protocol for a US Veteran with substance dependence and cooccurring severe mental illness. CM targeted attendance at outpatient appointments and appropriate use of hospital resources. Effects of CM were assessed by comparing the 3-month baseline and CM periods. The CM intervention marginally reduced unnecessary hospital admissions, resulting in cost savings to the medical center of over $5,000 in three months for this individual. However, CM did not affect outpatient attendance. Several complications arose, highlighting challenges in using CM in populations with substance use and cooccurring disorders. Practical suggestions are offered for maximizing the effects of CM.

A trap-nesting program in northern Spain undertaken in cooperation with Dr. Enrique Asensio (Instituto Nacional de Investigaciones Agrarias, Valladolid) over the past few years has resulted in numerous nests of megachilids. One of these represents a new species of Protosmia Ducke. It is described here in order to make the name available for the description of its nesting biology, and is named in honor of Dr. Asensio, in recognition of his considerable help in this trap-nesting study.In the following description, abdominal segments are numbered based on the metasoma; expressions in parentheses in the description of the male represent departures among male paratypes from characteristics described for the holotype.