Expression and characterisation of a Plasmodium falciparum protein containing domains homologous to sarcalumenin and a tyrosine kinase substrate, eps15.

We have identified in Plasmodium falciparum a novel gene encoding a putative bi-functional protein, termed PfPast-1, from genomic and cDNA libraries. Analysis indicated that the sequence encodes a 62 kDa protein of 529 amino acid residues with two distinctive domains: a sarcalumenin-like domain of approximately 320 amino acids at the amino half of the molecule, which shares homology to a major sarcoplasmic reticulum lumenal protein, sarcalumenin, and an eps15 homology domain of about 90 amino acids located at the carboxyl terminus. The eps15 homology domain, first identified in a tyrosine kinase substrate, eps15, and found in increasing numbers of mammalian proteins, has recently been suggested as a protein-protein interaction domain involved in intracellular sorting. Genomic sequences encoding similar proteins containing both the sarcalumenin-like and eps15 homology domains have been identified in humans and Drosophila. RNA blot analysis revealed the presence of a single messenger RNA transcript approximately 3.7 kb in size, which is expressed in all the developmental stages examined with the highest level in extracellular gametes followed by erythrocytic asexual stages, and the lowest in the gametocytes. In the attempt to define its biological function, we have expressed a full-length recombinant PfPast-1 protein in Escherichia coli. Specific immune serum directed against the recombinant protein recognised a approximately 55 kDa protein in the parasite lysate. Further characterisation of PfPast-1 may help in elucidation of its functions in P. falciparum.

Hydroxyurea-induced synchrony of DNA replication in the Kinetoplastida.

We have developed a reliable and reproducible method to induce synchrony of the DNA synthetic cycle in the Kinetoplastida. The method involves treatment of cultures with 20 mM hydroxyurea (HU) and fetal bovine serum. Both stationary-phase and exponential-phase cultures can be synchronized. However, in the case of exponential-phase cultures the population doubling time and rate of DNA synthesis of the population influenced the time of exposure to HU. The treatment of kinetoplastids with 20 mM HU did not adversely affect the cells as judged by oxygen consumption, RNA, and protein content. We postulate that the requirement for high HU levels, which would be toxic to vertebrate cells, may be due to a lower affinity of kinetoplastid ribonucleotide reductase, the target enzyme for HU. Some of the kinetoplastids are pathogens of man and his food chain. Consequently, the development of a reliable technique for synchronization of the kinetoplastids should not only permit a detailed analysis of their cellular and molecular biology but provide a means to collect and characterize biochemical and immunochemical substances relevant to the infectious process.

Identification, isolation, and characterization of naturally-occurring Trypanosoma cruzi variants.

Naturally occurring DNA variants of the single-cell-derived Y-02 stock of Trypanosoma cruzi were discovered during a routine assay of the stock. Three DNA variant types were isolated. One type was indistinguishable from the parental Y-02 stock on the basis of total DNA cell-1. The other two types contained approximately 30% and 70% more DNA cell-1 than the parental Y-02 stock. Both the nucleus and kinetoplast were involved in the DNA content differences. The increase in DNA cell-1 was not G-C- or A-T-specific and was unrelated to the developmental stage of the parasite. Epimastigote population doubling times, isoenzymes, and schizodeme analyses could not differentiate the variant stocks. However, marked karyotype polymorphisms were observed by pulse-field gel electrophoresis, and restriction-fragment-length-polymorphisms were detected in hybridizations of some endonuclease-restricted samples to the spliced leader probe. We postulate that the Y-02 variants are genetic homologs. The ability to form viable hybrids or aneuploids provides T. cruzi with a mechanism to survive environmental stress, promote intra-specific heterogeneity and generate the diversity observed in the presentation and course of Chagas' disease.

An instructional module concerning oral implantology: II. Development and implementation.

This paper represents the second of a series of papers documenting the design/development process of an individualized instructional module concerning oral implantology. This module was specifically designed for predoctoral dental students. The first paper discussed the rationale for the module and the initial field testing of the preliminary module. This paper examines the design and development of the oral implantology instructional module given to second and third year predoctoral dental students at the Medical College of Georgia School of Dentistry.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of Trypanosoma cruzi clones in inbred mice. III. Histopathological and electrocardiographical responses to chronic infection.

Histopathological and electrocardiographical (ECG) changes occur in the heart of C3H/HeN and C57BL/6 mice infected for 1 year with Trypanosoma cruzi clones Sylvio-X10/4 (X10/4), Miranda/78 (M/78), or Miranda/80 (M/80). Heart parasitism and a variable degree of inflammation occurred following infection with clones X10/4 or M/78 but not with M/80. Clone X10/4 caused more extensive myocardial inflammation and fibrosis than clone M/78. Myocardial fibrosis was more extensive in C3H than in C57 mice infected with clone X10/4. The normal ECG pattern of C3H mice is distinctly different from C57 mice. The PR intervals of mice infected with clone X10/4 greater than M/78 greater than M/80 approximately equal to controls. ECG abnormalities occurred more frequently in mice infected with clone X10/4 than in controls or mice infected with either M/78 or M/80 regardless of strain or sex and were generally more severe in C57 than in C3H infected with X10/4. First degree atrioventricular block occurred more frequently in C3H mice infected with clone X10/4 or M/78 and C57 mice infected with X10/4 than in all other groups. Complete atrioventricular dissociation occurred frequently in C57 mice infected with X10/4 and rarely in other mice. These results demonstrate that the myocardial response of mice to T. cruzi infection, both histological and electrophysiological, is modulated by both the mouse strain and the parasite isolate used.

Flow cytometric analysis of the DNA synthetic cycle of Candida species.

The total DNA per cell and DNA synthetic cycle phases were determined by flow cytometry in five Candida isolates including three species: Candida albicans 208R1, Candida tropicalis ATCC 750, and Candida parapsilosis 970, 3138, and ATCC 22019. The cells were prepared for flow cytometry by fixation in Carnoy fixative followed by staining with mithramycin. Marked but stable and reproducible inter- and intraspecific differences in total DNA per cell of stationary-phase cultures were found which did not correlate directly to diphenylamine estimates of the same parameter. This discrepancy was resolved by mathematically converting flow cytometry data into diphenylamine data. The reason for the discrepancy was found in studies of the DNA synthetic cycle of these yeasts: a large but isolate-specific variable proportion of the population is arrested in the S and G2-M phases after the culture passes from exponential to stationary phase. Histograms of exponential-growth-phase Candida isolates demonstrate that the majority of the population is in the G2-M phase of the DNA synthetic cycle. The DNA content of the C. tropicalis and C. parapsilosis isolates studied is as high as or higher than that of C. albicans. Extranuclear fluorescent particles were observed in the C. tropicalis isolate. No equivalent particles could be detected in the other four Candida isolates. The nature of the particles is unknown.

Comparative studies of the infection of Lewis rats with four Trypanosoma cruzi clones.

Quantitative and qualitative differences in parasitaemia levels and histopathological characteristics occurred in Lewis rats infected with four Trypanosoma cruzi clones. Rats infected with clone Sylvio-X10/7 sustained high parasitaemias and died during the acute phase whereas rats infected with clones Sylvio-X10/4, Miranda/78 or Miranda/80 did not. Myocardial fibrosis was more extensive in rats chronically infected with clone X10/4 than in rats infected with the 2 Miranda clones. Myocardial fibrosis developed more rapidly in rats than has been reported in mice. These characteristics are advantageous in developing animal models of human chagasic myocardiopathy.

Identification and analysis of epimastigote surface and metabolic proteins in Trypanosoma cruzi.

Metabolic and surface membrane proteins of four epimastigote-stage Trypanosoma cruzi clones were analyzed by one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No major inter-clonal differences were observed in the metabolic protein patterns, indicating that these proteins are highly conserved. However, marked quantitative and qualitative differences were observed in surface-labeled protein patterns following both one and two-dimensional electrophoretic analyses. Inter and intra-clonal differences in antigenic properties also were demonstrated by immunoprecipitation of the surface proteins with sera from animals immunized or infected with various T. cruzi stocks. Thus, a wide spectrum of both phenotypic and antigenic diversity exists in T. cruzi which may be relevant to problems of the diagnosis and immunotherapy of Chagas' disease.

Charity care: a proposal for reform.

Changing political attitudes and new financing trends jeopardize our commitment to providing equitable access to medical services for all citizens regardless of ability to pay. The commitment is further threatened by the lack of coordination and the fragmentation that make charity care expensive and ineffective. This article proposes that we bring continuity and consistency to our charity care efforts by considering four options for reform: reallocate health spending to emphasize primary, preventive care in the community setting: create special HMOs for the medically disadvantaged through a revitalized community health center program; develop a voucher program to ensure access to the HMOs as well as risk-sharing pools to ensure access to secondary and tertiary care; and radically restructure the public hospital systems and eliminate most hospitals in the veterans administration system.

A histopathological analysis of the course of myocarditis in C3H/He mice infected with Trypanosoma cruzi clone Sylvio-X10/4.

The histopathology of hearts of C3H/He (MTV-) mice infected for 2 to 440 days with Trypanosoma cruzi clone Sylvio-X10/4 was examined. Inflammation was present in most hearts at 14 days post-infection (d.p.i.) and persisted thereafter. Myocardial fibrosis, which was first seen 45 d.p.i. and was more marked after 200 d.p.i., developed at a time when few parasites were present. Fibrosis predominated in subepicardial and subendocardial myocardium, areas of most severe inflammation. Parasites were restricted to myocardial fibres and were found in all mice infected for 14 to 30 days and, in fewer numbers, in 54% of mice infected for more than 30 days. T. cruzi-induced myocarditis in mice was not a self-resolving disease but a dynamic, active process occurring continuously over a long time following infection. This clone-derived T. cruzi stock produced a myocarditis in inbred mice similar to that seen in human chronic Chagas' disease.

Trypanosoma cruzi: constancy of clone pathogenicity for inbred mice during long-term in vitro maintenance.

The reproducibility of infection of C3H/He mice with T. cruzi clones Sylvio-X10/4 and Sylvio-X10/7 maintained in the laboratory for 946 and 496 days respectively was assayed. Clone X10/7 from 15 different in vitro passages consistently induced an acute lethal infection (94.3% mortality) and constant survival time (Mean = 24.6 d.p.i.). Female mice survived significantly longer than males and mice older than 30 days at the time of infection survived significantly longer than younger mice. The mortality of mice infected with clone X10/4 from 23 different in vitro passages was lower (5.1%) and their survival longer (Mean = 42.7 d.p.i.) than mice infected with clone X10/7. High anti-T. cruzi IgG titres were detected in the plasma of all mice killed after 30 d.p.i. Histologically, the heart, skeletal muscles and/or large intestine contained intracellular parasites in 59% of the mice; parasites were found in mice of all groups tested. The hearts of all mice were comparably inflammed regardless of parasite passage number or duration of infection. These data demonstrate that the presentation and course of infection of inbred mice with two T. cruzi clones is not changed by either the duration or protocol used for in vitro maintenance; the reported loss or change in virulence of T. cruzi strains during long-term in vitro maintenance did not occur. Consequently, T. cruzi clones and inbred mice provide a dependable and reproducible means to study host-parasite interactions.

Studies of Trypanosoma cruzi clones in inbred mice. II. Course of infection of C57BL/6 mice with single-cell-isolated stocks.

A study of the course of parasitemia and mortality of C57BL/6 mice infected with the Trypanosoma cruzi Sylvio-X10 strain and two single-cell-isolate clones of the strain confirms our previous report on the existence of intra-strain subpopulations differing in their pathogenicity for inbred mice. In contrast to the generally accepted pattern of mouse strain susceptibility, C57BL/6 mice are more susceptible than C3H/HeN mice when infected with the Sylvio-X10 isolates. In addition, sex-related differences in susceptibility occurred depending upon the strain of the mouse and parasite isolate used. These data infer an interplay of host and parasite genetic factors influencing the outcome of a mouse infection with T. cruzi.

Studies of Trypanosoma cruzi clones in inbred mice. I. A comparison of the course of infection of C3H/HEN- mice with two clones isolated from a common source.

Two single-cell-isolate cloned stocks of the Sylvio-X10 strain, recovered from an acute human Trypanosoma cruzi infection, were used to infect C3H/HEN mice. The Sylvio-X10/4 clone produced a chronic infection in mice; clone Sylvio-X10/7 produced an acute lethal infection under identical experimental conditions. The course of infection of mice with the Sylvio-X10/7 clone was characterized by higher peripheral blood parasitemia and greater tissue involvement, an earlier appearance of specific anti-T. cruzi plasma IgG and shorter survival times than in mice infected with the Sylvio-X10/4 clone. The course of infection in mice with the Sylvio-X10 strain was intermediate between that of the two clones. This is the first demonstration of the pluripotential pathogenetic nature of a T. cruzi strain due to genetic heterogeneity of the population of parasites that constitute the strain. This experimental system is highly stable and reproducible. Consequently, the use of inbred mice and T. cruzi clones appears to provide an excellent model to study factors which influence the course of Chagas' disease.

Trypanosoma cruzi: flow cytometric analysis. I. Analysis of total DNA/organism by means of mithramycin-induced fluorescence.

Total DNA/organism was determined by flow cytometry on stocks of 33 single-cell-isolate clones and one strain of mithramycin-stained Trypanosoma cruzi. Interstrain differences in mean total DNA/group of 34% and interclone differences in total DNA/organism of 41% were found. Microspectrofluorometric analyses of the trypomastigote stage of selected clones confirmed the flow cytometry data and indicated that the total DNA/organism differences were due to differences in DNA of both the nucleus and kinetoplast with the nucleus being the major contributing factor. These data imply that the potential for genetic diversity in T. cruzi may be very large.

We have seen rate review, and it works.

In Maryland, state rate review is successful because of cooperation at all levels. Although there was friction in the beginning between the state commission and health care providers, the latter, with strong encouragement from their associations, played an important role in adopting a constructive program.

Maryland Health Care System, Inc.: a creative response to change.

Maryland Health Care System, Inc. represents the first multihospital activity in a large eastern city to incorporate a subsidiary management system. The creative coordination of health care capabilities encouraged by the MHCS interpretation may be applied elsewhere as new hospital systems develop.