Novel kinase platform for the validation of the anti-tubercular activities of Pelargonium sidoides (Geraniaceae).

BACKGROUND: Pelargonium sidoides is an important traditional medicine in South Africa with a well-defined history of both traditional and documented use of an aqueous-ethanolic formulation of the roots of P. sidoides (EPs 7630), which is successfully employed for the treatment of respiratory tract infections. There is also historical evidence of use in the treatment of tuberculosis. The aim of this study was to develop a platform of Mycobacterium tuberculosis (Mtb) kinase enzymes that may be used for the identification of therapeutically relevant ethnobotanical extracts that will allow drug target identification, as well as the subsequent isolation of the active compounds. RESULTS: Mtb kinases, Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase were cloned, produced in Escherichia coli and characterized. HPLC-based assays were used to determine the enzyme activities and subsequently the inhibitory potentials of varying concentrations of a P. sidoides extract against the produced enzymes. The enzyme activity assays indicated that these enzymes were active at low ATP concentrations. The 50% inhibitory concentration (IC50) of an aqueous root extract of P. sidoides against the kinases indicated SK has an IC50 of 1.2 µg/ml and GK 1.4 µg/ml. These enzyme targets were further assessed for compound identification from the P. sidoides literature. CONCLUSION: This study suggests P. sidoides is potentially a source of anti-tubercular compounds and the Mtb kinase platform has significant potential as a tool for the subsequent screening of P. sidoides extracts and plant extracts in general, for compound identification and elaboration by selected extract target inhibitor profiling.

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

Production of the Aspergillus aculeatus endo-1,4-beta-mannanase in A. niger.

The beta-mannanase gene (man1) from Aspergillus aculeatus MRC11624 (Izuka) was patented for application in the coffee industry. For production of the enzyme, the gene was originally cloned and expressed in Saccharomyces cerevisiae. However the level of production was found to be economically unfeasible. Here we report a 13-fold increase in enzyme production through the successful expression of beta-mannanase of Aspergillus aculeatus MRC11624 in Aspergillus niger under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpd (P)) and the A. awamori glucoamylase terminator (glaA(T)). The effect of medium composition on mannanase production was evaluated, and it was found that the glucose concentration and the organic nitrogen source had an effect on both the volumetric enzyme activity and the specific enzyme activity. The highest mannanase activity levels of 16,596 nkat ml(-1) and 574 nkat mg(-1) dcw were obtained for A. niger D15[man1] when cultivated in a process-viable medium containing corn steep liquor as the organic nitrogen source and high glucose concentrations.

Knowledge is payment: understanding state prompt-payment laws.

Faced with lagging receivables, hospitals across the country increasingly are suing delinquent payers to obtain payment. States have responded to the provider payment crisis by enacting and enforcing a rash of new laws requiring payers to pay claims within strict time frames or face steep penalties. To take advantage of the protection afforded by these laws, providers need to be aware of prompt-payment statutes in states in which they provide care, in which their patients live, and in which their payers are located. By becoming aware of their rights and obligations under these laws, providers can use the prompt-payment regulations proactively to avoid payment backlogs without resorting to litigation.

Malawi: the Peace Corps challenges.

PIP: An acquired immunodeficiency syndrome (AIDS) prevention project was begun in Malawi in January 1993 by Peace Corps Malawi. 23 workers strive: 1) to offer health education and counseling with regard to human immunodeficiency virus (HIV) infection and AIDS in district hospitals, health centers, and health clinics; 2) to encourage the participation of community groups in AIDS prevention, education, and counseling support activities; and 3) to implement AIDS education in primary and secondary schools. Volunteer activities include health education, home-based care, school programs (drama groups, peer counseling), income-generating activities, condom distribution, and formation of district committees and sub-committees. Target groups include women and youth. Sexual health is promoted. Malawian counterparts are being trained by the volunteers to ensure sustainability; local volunteers are becoming important as resources become scarce. The program is becoming decentralized as it moves into the villages, and community groups are increasing in number. Obstacles include: 1) the lack of resources; 2) the fact that the District AIDS Coordinators are also clinical officers and medical assistants and so can serve only part time; 3) the cultural taboos that make discussion of certain topics difficult; 4) the political system; 5) illiteracy among women; 6) drug abuse among youth; and 7) the difficulty of remaining separated from one's work when one is surrounded by clients, including coworkers, who are HIV positive.^ieng

The government's best offense is deference: the decision of the Supreme Court in Shalala v. Guernsey Memorial Hospital.

The United States Supreme Court agreed with the Secretary of Health and Human Services that Guernsey Memorial Hospital's advance refunding transaction costs would be subject to a medicare reimbursement policy that is not based upon generally accepted accounting principles. According to the sharp dissent in this case, this policy, set forth in a manual provision, contradicts federal regulations.

Medicare and GAAP: understanding the decision of the Sixth Circuit in Guernsey Memorial Hospital v. Secretary of Health and Human Services.

The Medicare Act entitles qualified providers to reimbursement for the "reasonable cost" of furnishing hospital services to Medicare beneficiaries. The Sixth Circuit's decision in Guernsey Memorial Hospital demonstrates the tension between generally accepted accounting principles and Medicare reimbursement principles.

Analysis of an incompetence case.

In recent months various Maryland health occupational boards have come under fire for their response to complaints against particular licensees. While this criticism may or may not be justified, the issue of how to proceed against incompetent licensees is a common problem nationwide. In Maryland's explosion of cases involving malpractice, far more licensees are disciplined for offenses relating to drugs or sex than for incompetence. An approach to make the boards more responsive to the problem of the incompetent licensee is presented.

Serial exercise testing after coronary artery bypass surgery.

To determine the duration of functional benefit from coronary bypass surgery, 111 patients with angina pectoris were serially evaluated by standard exercise testing prior to and for up to 4 years after surgery. Exercise testing 6 to 18 months after surgery showed greater heart rate-blood pressure product at peak work load, improved work capacity, and less symptomatic and electrocardiographic evidence of ischemia than was demonstrated preoperatively. Twenty patients were tested 37 to 48 months postoperatively and showed improved exercise performance in comparison with preoperative results, but the frequency of positive tests during this period no longer differed. Thus, improved exercise performance appears to persist for at least 4 years after coronary bypass surgery.

Expulsion of Nippostrongylus brasiliensis by mice deficient in mast cells.

Expulsion of the intestinal helminth, Nippostrongylus brasiliensis, occurs spontaneously about 2 weeks after a primary infection of rats and mice. Cellular changes in the small intestine coincident with the period of expulsion have suggested several mechanisms by which this 'self-cure' may be effected. Local anaphylaxis was proposed as a possible means of parasite clearance; this hypothesis has been supported by the demonstration of specific reaginic antibody production and jejunal mast cell accumulation in infected animals. In addition, increased mucus secretion and more recently, goblet cell proliferation in the jejunal mucosa of rats have been noted and considered as potentially important in mediating the self-cure reaction. The data presented below indicate that in the absence of demonstrable mast cells, the course of a primary infection with this parasite is unchanged; however, they are supportive of a role for globlet cells in the self-cure reaction.

Decussion geometrics in the goldfish nervous system: correlation with probability of survival.

In the goldfish, the optic nerve decussation occurs without intermingling of fibers from the two eyes. In two-thirds of juvenile and adult specimens, the left optic nerve is dorsal at the midline. In about 60% of the specimens, the decussation of Mauthner's neuron also has a left-dorsal-to-right (L/R) configuration. Concordance for decussation geometry is greater than 80%, with smaller specimens accounting for a disproportionate number of discordant cases. In embryos and very young larvae, the L/R configuration occurs in slightly less than 50% of optic chiasmata and in slightly more than 50% of Mauthner's cell chiasmata, and there is no significant tendency toward concordance. However, larval specimens that survive 1 month after hatching are markedly skewed toward adult decussation patterns and somewhat skewed toward concordance. These observations, together with results of cannibalization and predation experiments, suggest that the adult population pattern can be achieved through selection pressures against discordant individuals and, perhaps separately, against individuals with right-dorsal-to-left (R/L) chiasmatic configurations. Decussation patterns correlate with embryonic postures, larval orientation preferences, and growth rates after hatching, which may themselves serve as a basis for selection.

Histological evidence for direct connections between the optic lobes of the cockroach Leucophaea maderae.

Heretofore, descriptions of direct interconnections between insect optic lobes have been based on histological examinations of normal brains or on inference from electrophysiological or behavioral data. We present here what we believe to be the first demonstration of such monosynaptic connections by techniques of experimental neuroanatomy. Twenty-four to 39 h after extirpation of the left optic lobe, degenerating axons and axon terminals, as silvered by a modified Nauta technique, were abundant in the central portion of the medulla of the right optic lobe. The periphery of the medulla was free of argyrophilic debris as were the lobula and lamina. The distribution of neuronal somata with processes terminating in the the left optic lobe was established by retrograde axonal transport of horseradish peroxidase injected into the left lobe and by the development of distinctive perinuclear rings of RNA (a 'chromatolytic' reaction) by some cells within 1-2 weeks following amputation of the left lobe. Both techniques revealed distinct clusters of cells in the anteroventral and posterior regions of the right optic lobe, and in the medial portion of the right protocerebrum. The cells which interconnect the two optic lobes may be involved not only in the bilateral representation of visual information, but also in the coordination of optic lobe pacemakers which control a circadian rhythm of locomotory activity.