Willingness to pay for voluntary health insurance in Tanzania.

OBJECTIVE(S): To assess how willing people would be to join a voluntary health insurance scheme and to see how they respond to changes in the benefit package. We also examined willingness to cross-subsidise the poor. DESIGN: Cross-sectional study. SUBJECTS: Two thousand two hundread and twenty four households comprising of 1,163 uninsured household heads asked about their willingness to pay for insurance in seven districts/councils (three urban and four rural) and 1,061 insured households were asked about their willingness to pay for insurance premiums for the poor in their community. Uninsured respondents were presented with two scenarios, the first reflected the current design of the Community Health Fund/Tiba Kwa Kadi (CHF/TIKA), the second offered expanded benefits, and included inpatient care in public facilities and transport. RESULTS: Only 30% of uninsured rural households were willing to pay more than Tsh 5,000 the current premium level, their average amount was Tsh 10,741, while in urban areas one percent of households were willing to pay more than Tsh 5,000. There was very limited willingness to pay more than 5,000 Tsh, even with an expanded package in rural areas. Household from rural areas were more willing to cross-subsidise the poor, but contribution levels were higher in urban areas. CONCLUSION: Communities need to be sensitised about the existence of the CHF/TIKA to encourage enrollment. Expanding the benefit package would further increase enrollment. However, few people would be willing to pay more than the current premium.

Localization of human coagulation factor VIII (hFVIII) in transgenic rabbit by FISH-TSA: identification of transgene copy number and transmission to the next generation.

For chromosomal localization of the hFVIII human transgene in F2 and F3 generation of transgenic rabbits, FISH-TSA was applied. A short cDNA probe (1250 bp) targeted chromosomes 3, 7, 8, 9 and 18 of an F2 male (animal 1-3-8). Two transgenic offspring (F3) revealed signal positions in chromosome 3 and chromosomes 3 and 7, respectively. Sequencing and structure analysis of the rabbit orthologous gene revealed high similarity to its human counterpart. Part of the sequenced cDNA (1310 bp) served as a probe for FISH-TSA analysis. The rabbit gene was localized in the q arm terminus of the X chromosome. This result is in agreement with reciprocal chromosome painting between the rabbit and the human. The presented FISH-TSA method provides strong signals without any interspecies reactivity.

Localization of the single copy gene Mdh2 on Xenopus tropicalis chromosomes by FISH-TSA.

A single copy gene, mitochondrial malate dehydrogenase 2 (Mdh2), was localized on Xenopus tropicalis chromosomes by fluorescence in situ hybridization coupled with tyramide signal amplification (FISH-TSA). The respective cDNA was cloned and sequenced. The labeled probe hybridized with a subcentromeric region of the long arms of homologous chromosomes 3. Results of comparison of the gene localization with previously mapped X. laevis paralogs strongly suggest a common evolutionary origin of chromosomes 3 and 8 in X. laevis and chromosome 3 in X. tropicalis. This is the first time that a single copy gene has been visualized on X. tropicalis chromosomes. The FISH-TSA method gives a strong signal with a 1-kb labeled probe.

Xstir polymorphism and absence of sex linkage in Xenopus laevis ME2 gene.

A fragment of ME2 cDNA from exon 2 to exon 11 was sequenced and the sequence submitted to GenBank. Analysis of the intron, probably intron 13, revealed a polymorphism which is due to the presence of tandem repetitions of Xstir elements. Genetic analysis of the parents and the offspring showed a standard distribution of intron variants. This distribution was not dependent on sex. We conclude, contrary to previous reports, that the ME2 gene is not linked to sex. Consequently, the Xstir polymorphism can be used as a tool for genetic analysis.

The c-SRC1 gene visualized by in situ hybridization on Xenopus laevis chromosomes.

Fluorescent in situ hybridization followed by tyramide signal amplification was used to map the site of the c-SRC1 gene on XENOPUS LAEVIS chromosomes. Positive results were obtained with a cDNA probe of about 1 kb. The c-SRC1 gene is located in the subcentromeric region in the long arm of one of the acrocentric chromosomes of the G category (classified according to Graf and Kobel, 1991). The c-SRC1 gene and the XENOPUS major histocompatibility complex (MHC) 1b locus, which consists of 20 tandemly arranged gene copies, are situated on different chromosomes of the G category.

Genomic organization of the mouse src gene. Sequencing of src introns revealed a new chromosome 2 microsatellite marker.

Ten introns interrupting the coding sequence of the mouse src protooncogene were sequenced (in total 11260 bp) and their general characteristics compared with the homologous genes in human and chicken. While the study of genome organization of the src gene was performed only in the inbred mouse strain BALB/cHeA (Mus musculus domesticus), one special region in the intron 5 was also sequenced in additional mouse strains (M. musculus musculus and M. spretus), because the discovered CA and GT repeat array differences could serve as a new polymorphic marker in the chromosome No. 2 and help elucidate some evolutionary relations between mouse strains.

Development of transgenic Xenopus laevis with a high C-src gene expression.

Xenopus laevis larvae with an elevated expression of c-src were generated by mating a transgenic X. laevis male frog carrying proviral Rous sarcoma virus (RSV) long terminal repeat (LTR) and most of the pol gene sequences in its sperm DNA and a normal X. laevis female frog. Offspring (15-20%) with a higher dosage of c-Src, detected in disorganized myotomal musculature and in cerebral and spinal neuronal cells by immunohistochemical analysis, developed abnormally, with edemas (in most cases), head deformities, and eye and axial system defects. In the remaining embryos, a small increase in c-src expression seemed to be compatible with normal embryogenesis. The dosage of c-Src correlated with the dosage of RSV LTR integrated in frog DNA as revealed by Southern and polymerase chain reaction (PCR) analyses. Authenticity of the integrated RSV LTR including enhancer sequence was proved by sequencing. Probing of total RNA from aberrant larvae demonstrated several times higher dosage of c-src mRNA in their tissues than in control tadpoles. We hypothesize that the integrated RSV regulatory sequences can stimulate the expression of c-src proto-oncogene of X. laevis above a threshold that interferes with the early developmental program of frog embryos.

Uptake of plasmid RSV DNA by frog and mouse spermatozoa.

Xenopus laevis spermatozoa and mouse epididymal spermatozoa were compared in their ability to bind plasmid DNA. Spermatozoa of both species are endowed with a very similar binding capacity for plasmid pAPrC DNA carrying the complete Rous sarcoma virus (RSV) DNA. Our experiments failed to detect any substantial differences between both types of sperm cells in the kinetics of DNA binding, maximum DNA binding capacity, effect of various ions, metabolic inhibitors and substrates on DNA binding, etc. Each X. laevis and mouse sperm cell associates, on an average, with about 50 and 45 molecules, respectively, of the plasmid DNA in a DNase resistant form, if spermatozoa were exposed to the DNA at a concentration of 0.5 microgram/5 x 10(6) sperm cells. The uptake of the DNA by both types of sperm cells is strongly inhibited by heparin. The 37-kDa factor IF-1 shown by Zani et al. (1995) to specifically block DNA binding to mouse sperm cells inhibited the interaction between pAPrC DNA and frog spermatozoa with a similar intensity.

Association of rous sarcoma virus DNA with Xenopus laevis spermatozoa and its transfer to ova through fertilization.

Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70-160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0-1.4 micrograms/10(7) sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25-30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed.

Autoantibodies against histones and actin in patients with rheumatic diseases assessed by the western blot method.

Using the Western blot method, the authors analyzed 85 sera obtained from patients with rheumatic diseases, focused on the presence of antihistones and antiactin autoantibodies. The authors detected a 32% incidence of the two investigated autoantibody specificities. In a group of 42 patients with systemic lupus erythematosus in 22 sera (52%) positive antihistone antibodies were present, whereby autoantibodies anti-H1 and anti-H2B were most frequent. In 15 sera in this group of patients (36%) antiactin autoantibodies were present.

[Autoantibodies against histones and actin determined by western blotting in patients with rheumatic diseases]. / Autoprotilátky proti histonum a aktinu u nemocných s revmatickými onemocneními urcené metodou western blotu.

Using the Western blot method, the authors analyzed 85 sera obtained from patients with rheumatic diseases, focused on the presence of antihistones and antiactin autoantibodies. The authors detected a 32% incidence of the two investigated autoantibody specificities. In a group of 42 patients with systemic lupus erythematosus in 22 sera (52%) positive antihistone antibodies were present, whereby autoantibodies anti-H1 and anti-H2B were most frequent. In 15 sera in this group of patients (36%) antiactin autoantibodies were present.

Transbilayer redistribution of phosphatidylserine in erythrocytes of a patient with autoerythrocyte sensitization syndrome (psychogenic purpura).

We have investigated a female patient with autoerythrocyte sensitization syndrome (AES syndrome), having a positive skin response to her own red blood cells (RBC) and to phosphatidylserine (PS). Using 2,4,6-trinitrobenzenesulfonic acid (TNBS), bee venom phospholipase A2 and merocyanine 540 binding, we have demonstrated that in RBC of patient more than 50% of PS is redistributed into the outer leaflet of the plasma membrane. Using homologous RBC from a healthy donor we were able to induce transbilayer PS redistribution by incubation with the patient plasma. The presence of immunoglobulin E against cardiolipin and PS was proved in patient's plasma. We elaborated a method for cytoskeleton visualization using indirect immunofluorescence technique. We found disorders in cytoskeleton organization in RBC of the patient. We recommend in vitro testing for AES syndrome diagnosis. The positive effect of chlorpromazine treatment is described.

Isolation and characterization of a lectin from Sarcocystis gigantea.

A hydrophobic, galactose-specific lectin was isolated by means of affinity chromatography from Sarcocystis gigantea. Adsorbents with different spacer lengths were tested. S. gigantea lectin differs from sheep muscle lectin in the spacer length needed for adsorption. Sodium dodecyl sulfate-gel electrophoresis of the S. gigantea lectin revealed a subunit size about 19 kDa and the presence of disulfide cross-linked dimers. The lectin is present in high concentration in cystozoites, cyst fluid and cyst wall material.

An immunocytochemical method for the visualization of tubulin-containing structures in the egg of Xenopus laevis.

Tubulin can be isolated and purified from Xenopus laevis eggs through modification of Olmstedt's (1970) tubulin isolation method, viz. by repeating the vinblastin precipitation step after resuspension of the sediment in a detergent-containing stabilizing medium. By this we overcome the deleterious influence of the yolk granules in the isolation procedure. From 11 of Xenopus laevis eggs 25 mg VB-paracrystals can be obtained. The apparent molecular weight of the purified tubulin is 52,800. Antiserum against the purified Xenopus VB-paracrystals, raised in 2 Chinchilla rabbits, cross-reacts in immunodiffusion tests in agar gels with rat brain tubulin and with tubulin isolated from Xenopus laevis eggs by the described procedure. Specific indirect fluorescence staining and appropriate control reactions reveal that cilia of Tetrahymena pyriformis, cytoplasmic networks in cultured mouse Leydig cells, as well as mitotic spindles and nuclear regions in paraffin sections of Xenopus laevis blastulae, react with the antibodies against Xenopus laevis egg tubulin as well as with monoclonal antibodies against pig brain tubulin. These results provide additional evidence for the view that tubulin antibodies are neither species nor tissue specific and show that under appropriate conditions tubulin containing structures can be visualized in paraffin sections.

Biochemistry of metalocenes. The organ distribution of hydroxyacetyl [103Ru]ruthenocene and its glucuronide in mice.

Hydroxyacetyl [103Ru]ruthenocene and its o-glucuronide were prepared in vitro by incubation of acetyl [103Ru]ruthenocene with rat-liver homogenate, NADPH, and UDP-glucuronate. The factors affecting hydroxylation and glucuronidation in vitro were optimized for acetylruthenocene. Hydroxyacetyl [103Ru]ruthenocene glucuronide showed no affinity for the adrenal glands, but after i.v. administration of hy droxyacetyl [103Ru]ruthenocene there was a distinct accumulation of Ru-103 in adrenals, similar to that found after administration of acetyl [103Ru]ruthenocene.

Effect of native and modified bull seminal ribonuclease on tumour and testicular cells and phytohaemagglutinin-stimulated pig lymphocytes.

The carboxymethylated, oxidized and reduced forms of AS RNase inhibited transplantability and DNA synthesis of tumour cells BP-8 and EL-4 incubated in vitro. With tumour cells EL-4 the results under in vitro conditions did not not correspond to those obtained under the conditions in vivo. The survival of mice given injections of EL-4 cells and of the native and carboxymethylated AS RNase was only slightly prolonged. Mice that received intra-abdominally BP-8 cells and both carboxymethylated and oxidized and reduced forms of AS RNase survived two or three times longer than the controls. Succinylation and maleylation of AS RNase eliminated any antitumoral effect. Aspermatogenic activity of AS RNase was abolished by any modification of the molecule which had substantially reduced, or removed, the RNase activity. Neither native nor modified forms of AS RNase had an inhibitory effect on unstimulated pig lymphocytes. The DNA synthesis of PHA-stimulated lymphocytes was inhibited by the native and carboxymethylated AS RNase only. Bovine pancreatic A RNase had any inhibitory effect on neither tumour nor testicular cells.