Molecular characterization of the ldmdr1 multidrug resistance gene from Leishmania donovani.

The ldmdr1 gene that confers resistance to multiple structurally dissimilar hydrophobic drugs in Leishmania donovani has been isolated within a 5.4-kb XmaI fragment from a genomic library of L. donovani DNA and its protein coding region sequenced. The longest open reading frame within ldmdr1 encodes a 146.5-kDa protein of 1341 amino acids, designated LDMDR1. The primary structure and predicted membrane topology of LDMDR1 indicates that it is a member of the P-glycoprotein superfamily with the greatest homology to the mammalian multidrug resistance P-glycoproteins. A 2.3-kb SalI fragment derived from a second ldmdr1 allele was also cloned from the L. donovani library. Nucleotide sequence analysis of a portion of the SalI insert revealed 5 single base differences from its counterpart within the 5.4-kb XmaI fragment, one of which created a PvuI restriction site polymorphism. Southern blots of PvuI-digested DNA divulged that the amplified ldmdr1 gene copies in a multidrug-resistant L. donovani strain were all derived from the single ldmdr1 allele whose protein coding segment was sequenced in its entirety.

Molecular characterization of phosphoribosylpyrophosphate synthetase from Leishmania donovani.

The phosphoribosylpyrophosphate synthetase (PRS) enzyme from parasitic protozoa plays a critical role in the acquisition of exogenous purine bases by providing the phosphoribosylpyrophosphate substrate for phosphoribosylation. To characterize a PRS enzyme from parasitic protozoa, the prs gene was isolated from a genomic library of Leishmania donovani DNA. A 1936-bp SalI fragment was sequenced that encompassed an open reading frame of 1113 nucleotides encoding a polypeptide of 371 amino acids and 40 787 Da. After gap alignment, the leishmanial PRS exhibited 40-42% amino acid identity with a variety of mammalian and prokaryotic PRSs. L. donovani PRS also contained an approx. 20-amino acid stretch that was highly homologous to the phosphoribosylpyrophosphate binding domains of mammalian phosphoribosyltransferase enzymes. Two prs-specific transcripts of 2.6 and 2.1 kb were detected by Northern analysis, and Southern blots of genomic DNA implied that the prs locus was not tandemly repeated in the L. donovani genome. PRS activity was detected in L. donovani extracts, and apparent Km values of approx. 30 microM and approx. 1 mM were calculated for ribose-5-phosphate and ATP, respectively. PRS was sensitive to inhibition by AMP and ADP but refractory to IMP, GMP, GTP, CTP, and UTP. The high apparent Km value of the parasite enzyme for ATP and its insensitivity to inhibition by many nucleotides suggested that kinetic differences between the L. donovani and human PRSs could provide an avenue for rational therapeutic manipulation of parasitic disease. The isolation of the L. donovani prs gene now provides an opportunity to genetically dissect the determinants responsible for the function and regulation of this indispensable enzyme of purine and pyrimidine metabolism in a genus of parasitic protozoa.

Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.

Evaluation of drug reviews.

Drug reviews appearing in Clinical Pharmacy, Drug Intelligence and Clinical Pharmacy (DICP), Drugs, and Pharmacotherapy from January 1982 through December 1984 were evaluated for number, duplication among journals, timeliness, scope, and format. The design of this study was primarily quantitative rather than qualitative. Pharmacotherapy published the most reviews (49), followed by Drugs (43), Clinical Pharmacy (37), and DICP (29). Drugs and Pharmacotherapy published the largest number of unique reviews (agents not reviewed by the other journals during the study period), while Pharmacotherapy and Clinical Pharmacy published the most reviews on newly marketed drugs. Reviews of four drugs (acyclovir, moxalactam, ranitidine, and trazodone) were compared in terms of major sections, terminology and format, bibliography, use of tables and figures, scope of evaluative comments, and review process. Reviews in Drugs consistently contained the most references and tables and provided the most detail. Information was most accessible in Drugs, followed by Pharmacotherapy. Drugs used the largest panel of reviewers. All of the journals provided evaluative comments, although the scope varied. Continuing-education credit is available for review articles in Clinical Pharmacy and DICP. In selecting one or more of these journals, individuals or institutions should compare their needs with regard to the timeliness, scope, and format of the review articles in each journal.

Evaluation of 516 cardiopulmonary resuscitation attempts.

All adult cardiopulmonary resuscitations attended by the pharmacy department at a 486-bed tertiary-care institution were analyzed over a 24-month period. Data describing patient demographics, drug and equipment use, and patient survival were collected on 516 consecutive adult arrests. These data were recorded on a report form by a pharmacy technician and were classified as cardiac, respiratory, trauma, or other. Trauma included arrests caused by motor-vehicle accidents and gunshot wounds, and other included arrests caused by anaphylaxis or seizures. The majority of arrests (70%) were classified as cardiac, 24% as respiratory, and 6% as other. Overall, 54.5% of the patients suffering from arrests were resuscitated successfully. There was an equal distribution of arrests throughout the day. The mean duration of the resuscitation efforts was 38 minutes with a trend toward greater patient survival when resuscitation efforts lasted less than 15 minutes. Arterial blood-gas determinations were made in 81% of the arrests, defibrillations in 40%, and pacemaker or chest tube insertion in less than 10%. Sodium bicarbonate was the most frequently administered medication, followed by calcium salts and atropine sulfate. Lidocaine was used in 83% of the cases requiring antiarrhythmic therapy. Pressor support was required in 44.6% of the cases; norepinephrine bitartrate was the first-line pressor agent. Drugs not categorized as essential according to the American Heart Association's Advanced Cardiac Life Support (ACLS) standards were administered infrequently. Hospitals may benefit from arrest data in assessing their equipment and supply needs, staffing patterns, and personnel training programs.

Human figure drawing size as a measure of self-esteem.

The relationship between self-esteem and human figure drawing size was investigated. Seventy-six adolescents completed the Rosenberg Self-Esteem Scale and drew the human figure. For males only. there was a significant linear relationship between self-esteem and width of human figure drawing and a significant curvilinear relationship between self-esteem and height and self-esteem and area of human figure drawing. The latter findings lend support to a hypothesis regarding the possibility among low self-esteem subjects of a bimodal distribution with respect to size.