Protein kinase C activities with different characteristics, including substrate specificity, from two human HL-60 leukemia cell variants.

To evaluate the role of protein kinase C in the cellular maturation processes induced by phorbol diesters, we examined the biochemical activity of protein kinase C from HL-205, a cell variant from the human promyelocytic HL-60 leukemia that is susceptible to differentiation induced by phorbol 12-myristate 13-acetate, and from HL-525, an HL-60 variant that is resistant to such an induction. The activities of protein kinase C from the two cell types differed in their requirements for the cofactors Ca2+ and lipids. These enzyme activities also differed in their abilities to phosphorylate protamine and a series of four oligopeptides. We suggest that the differences in vitro in the activities of protein kinase C between HL-205 and HL-525 cells, especially in their substrate specificity, are closely related to the different phosphorylation patterns induced in vivo by phorbol 12-myristate 13-acetate in these cells. We also suggest that these differences may be responsible for the different susceptibilities of the two cell types to maturation induced by phorbol diesters.

Specific protein phosphorylation in human promyelocytic HL-60 leukemia cells susceptible or resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate.

The pattern of protein phosphorylation induced by phorbol-12-myristate-13-acetate (PMA) was analyzed by two-dimensional gel electrophoresis in human HL-60 leukemia cells, which are susceptible to induction of cell differentiation by PMA, and in cells from an HL-60 cell variant designated R-94 that are resistant to such an induction. Protein phosphorylation was detected by observing either a rapid acid-directed charge shift of [35S]methionine-labeled protein or an increase in the amount of phosphate label in a 32P-labeled protein. The results indicated that PMA at 10(-7) M causes within 30 min after treatment the phosphorylation of at least ten different proteins in both the HL-60 and R-94 cells. Among these ten phosphorylated proteins, we identified a major cytoplasmic polypeptide (Mr approximately 64,000), a cytoskeletal protein (Mr approximately 56,000), a nonmuscle myosin light chain, and two proteins (Mr approximately 60,000 and 64,000) localized in or around the cell nucleus. Phosphoamino acid analysis of six of the ten phosphoproteins showed that they contain phosphoserine. None of these proteins contained phosphotyrosine or phosphothreonine. The R-94 cell variant was found to be capable of increased protein phosphorylation after PMA treatment; however, the level of phosphate incorporation reached only the level of the untreated HL-60 cells and thus fell far short of the level observed in the HL-60 cells after PMA treatment. It is suggested that the basis for the acquired resistance in R-94 cells towards induction of cell differentiation by PMA is a block in signal transmission involving phosphorylation of nuclear protein(s) following the binding of the inducer PMA to its receptor (protein kinase C).

Control of macrophage cell differentiation in human promyelocytic HL-60 leukemia cells by 1,25-dihydroxyvitamin D3 and phorbol-12-myristate-13-acetate.

Human promyelocytic leukemia cells (HL-60) were induced to differentiate into macrophage-like cells in a dose (3 X 10(-10) to 10(-7) M) and time (1 to 6 days)-dependent manner by 1,25-dihydroxyvitamin D3 and the tumor promoter, phorbol-12-myristate-13-acetate. Differentiation was determined by an increase in the percentage of morphologically mature cells, in lysozyme and nonspecific esterase activities, and in reactivity with the murine OKM1 monoclonal antibody. Two HL-60 cell variants, designated as R-80 and B-II, were also examined. R-80 cells, which are resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate, also exhibited resistance, although to a lesser degree, to induction of cell differentiation by 1,25-dihydroxyvitamin D3. The resistance to the action of the two compounds is presumably not due to similar binding sites for the two inducers, since 1,25-dihydroxyvitamin D3 was unable to compete for the phorbol diester binding sites as measured by [3H]phorbol-12,13-dibutyrate binding. B-II cells were resistant to induction of cell differentiation by 1,25-dihydroxyvitamin D3, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide. Two-dimensional electrophoretic analysis of HL-60 cell protein patterns indicated that treatment of the HL-60 cells with 1,25-dihydroxyvitamin D3, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide caused the cells to express various monocyte-macrophage and granulocyte marker proteins. None of the inducers caused a protein pattern identical to that of peripheral monocytes or granulocytes in the HL-60 cells, but the protein pattern of the HL-60 cells treated with 1,25-dihydroxyvitamin D3 was the closest to that of peripheral blood monocytes. These results indicate that 1,25-dihydroxyvitamin D3 induces in the HL-60 cells a phenotype that resembles, but is not identical to, that of peripheral monocytes-macrophages.

Hydatidosis and cysticercosis: the dynamics of transmission.

The infective pattern of taeniids in their intermediate hosts is determined by the complex interaction of parasite- and host-related factors. Many of these have been examined in this review, but particular emphasis has been placed on environmental factors that affect the free-living egg. While number, infectivity and distribution of eggs are among the important factors determining the infective pattern, the dynamics of this aspect of transmission have been previously neglected. The biotic potential of the large taeniid tapeworms is enormous. Eggs deposited on pasture are subjected to the microclimatic effects of the environment. They seem to tolerate a relatively wide temperature range. Heat down to -30 degrees C. However, they are rapidly killed by low humidity at all temperatures. Under more favourable conditions, they have finite life spans largely determined by the environmental temperature. At deposition, the egg population seems to be at various stages of maturity. Immature eggs appear to be able to mature under suitable environmental conditions and to become infective. They then age, and their infectivity to the intermediate host declines. At the population level, the changes in infectivity with time are determined by the relative stages of maturity within the egg population and the temperatures that it experiences. Under certain circumstances the infectivity of a given population may increase temporarily. The movements and defaecation habits of the definitive host determine the primary site of egg deposition. However, evidence is accumulating that considerable dispersion occurs almost immediately afterwards. Eggs have been shown to disperse up to 80 m within 10 days and there are indications that small numbers travel much further. Intermediate hosts, such as cattle and sheep, generally avoid grazing areas contaminated with faeces. Thus, dispersal enhances the chance of the eggs being ingested. The combination of high biotic potential and long-range dispersal means that individual infected definitive hosts can be responsible for infecting intermediate hosts over a very wide area. Although various possible agents have been suggested, the mechanisms responsible for disseminating eggs are still uncertain.(ABSTRACT TRUNCATED AT 400 WORDS)

Australasian contributions to an understanding of the epidemiology and control of hydatid disease caused by Echinococcus granulosus--past, present and future.

For several years after Echinococcus granulosus was introduced into Australasia from Europe, its life cycle was unknown. Before the end of the 19th century, however, Cystic Hydatid Disease (CHD) was recognized in humans as an important cause of death, although its magnitude was not quantified. During the first 50 years of the 20th century, the urban component declined, but CHD remained a serious source of ill-health. During the second half of the 20th century major research contributions have included: (i) differentiation of pastoral from wildlife Echinococcus and (ii) breaking the 'epidemiological code' of the family Taeniidae, thereby defining the conditions under which transmission occurs. Based on this research and concurrent surveys, it is concluded that the present geographical distribution of E. granulosus in domestic animals and CHD in the rural population is determined by climate; being uncommon where hot dry seasons continue beyond a few months. The parasite's range may be extended in its wildlife cycle through a highly susceptible definitive host--the dingo. The ease with which blowflies transfer taeniid eggs from dog faeces to animal and human foodstuffs implies that in Australasia, CHD can sometimes be regarded as foodborne. A perceived decline in autochthonus cases in the rural community during the second half of the 20th century may be accounted for by increased standards of living generated by economic advancement. Where a decline has not occurred or incidence is unusually high, CHD may be acting as an indicator of social disadvantage. Field trials and control programmes demonstrated that pastoral echinococcosis readily responded to intervention. Indeed, in both New Zealand and Tasmania, using different administrative structures and methods of funding, E. granulosus was driven from endemic towards extinction status. The really convincing result was the rapid decline in CHD in all age groups. The problem still to be solved is to identify methods for eliminating foci where high levels of CHD still exist but, continent- or even state-wide intervention may not be considered to be cost-effective using current tools. Here, mathematical modelling and cost-benefit analysis can be regarded as powerful methods to assist the decision-making process.

Further evidence for the long distance dispersal of taeniid eggs.

Autopsy of Soay Sheep on St. Kilda revealed the presence of cysticerci of Taenia hydatigena despite the absence of the definitive host on the island. Both the intensity of infection and the prevalence increased with age implying that the sheep did not acquire immunity to reinfection or superinfection. The sheep on average ingested approximately 2.4 eggs per annum. This is far below that expected if an infected dog had visited the island even on a single occasion. The data provide evidence that taeniid eggs are being transported against the prevailing wind by wildlife from at least the nearest inhabited land mass to St Kilda some 60 km distant.

Transmission dynamics of Echinococcus granulosus, Taenia hydatigena and Taenia ovis in sheep in Uruguay.

A base-line survey was carried out on the transmission dynamics of Echinococcus granulosus, Taenia hydatigena and Taenia ovis in sheep in the Department of Florida, Uruguay. Mean life expectancy at birth of the sheep population in this Department was estimated at 3.5 and 4.8 years for male and female sheep, respectively. Both intensity and prevalence of E. granulosus infection increased with age providing no evidence that immunity was acquired in the population. This revealed that E. granulosus was only relatively stable and in an equilibrium endemic steady state with an estimate of the basic reproduction ratio (Ro) of about 1.2. The mature larval population in sheep was over-dispersed with parameter k, from fitting a negative binomial, being 0.199 and 0.556 in 42- and 54-month-old sheep, respectively. The mean number of cysts increased by 1.05 cysts per year and the infection pressure was calculated as 0.174 infections per year, implying that each infection consisted of 693.6 eggs to produce 6.03 cysts. Neither intensity nor prevalence of infection with T. hydatigena or T. ovis increased with age. This indicated that immunity was acquired to reinfection by both parasites and they were in an equilibrium hyperenedemic steady state with an estimated Ro of 4.2-5.8. Viability and fertility of E. granulosus, and viability of T. hydatigena cysts in the older age groups were low. The effects of the different transmission levels of the parasites on potential control strategies are discussed.

Rates of reinfection with Echinococcus granulosus, Taenia hydatigena, taenia ovis and other cestodes in a rural dog population in Uruguay.

A survey was undertaken to determine both the prevalence of, and reinfection rates with Echinococcus granulosus and other cestodes in the Department of Florida, Uruguay. Baseline prevalence was determined in 303 rural dogs which then, in 4 groups, were re-examined 2, 4, 8 or 12 months later. Baseline prevalences for E. granulosus, Taenia hydatigena, Taenia ovis and Dipylidium caninum were 13.2, 13.9, 2.3 and 13.2%, respectively. The frequency distribution of E. granulosus was over-dispersed. Dogs in the population became infected with E. granulosus between 2 and 4 months after treatment (prevalences at 2, 4, 8 and 12 months were 0, 6.8, 18.6 and 27.9%, respectively). There was no indication that there was a predisposition of dogs to infection with the Odds Ratio being 1.0. Dogs were infected with T. hydatigena and D. caninum within 2 months and with T. ovis between 2 and 4 months after treatment. The implications of these different rates of reinfection in the dog population on anthelmintic control strategies against cystic echinococcosis are discussed.

p53 gene deletions in paraffin-preserved lymphoid tumors from irradiated mice.

Experiments were performed to measure deletions in the p53 gene in paraffin-embedded tissues (tumors and control) derived from mice exposed to gamma-rays or neutrons up to 28 years ago. Deletions in exons 1, 3, 4, 5, 6, 7 and 9 were monitored by PCR and Southern blotting techniques. The results of these experiments demonstrated p53 deletions in only 1/6 spontaneous tumors but in 5/6 gamma-ray-induced and 5/6 neutron-induced tumors. Exons deleted in tumors from gamma-ray exposed mice were similar to those deleted in tumors from neutron-exposed mice. They document differences in spectra of p53 deletions in comparing spontaneous radiation-induced tumors.

Detection of retinoblastoma gene deletions in spontaneous and radiation-induced mouse lung adenocarcinomas by polymerase chain reaction.

A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma gene using histological sections from radiation-induced and spontaneous tumors as the DNA source. Six mouse Rb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mouse Rb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (5.69 Gy 60Co gamma rays or 0.6 Gy JANUS neutrons, which have been found to have approximately equal radiobiological effectiveness) were analyzed for mouse Rb deletions. Tumors in 6 neutron-irradiated mice had no mouse Rb deletions. However, 1 of 6 tumors from gamma-irradiated mice (17%) and 6 of 18 spontaneous tumors from unirradiated mice (33%) showed a deletion in one or both mouse Rb alleles. All deletions detected were in the 5' region of the mouse Rb gene.

Modification of radiation response in mice by fractionated extracts of Panax ginseng.

A water-soluble extract of the root of Panax ginseng, a plant native to northeastern China, was fractionated into three components: carbohydrate, protein, and saponin fractions. The fractions obtained were tested for their ability to protect against the lethal effects of 60Co gamma irradiation in C3H mice. The results were compared to the protective ability of the water-soluble fraction of whole ginseng. An experiment designed to test the optimum time of injection of whole ginseng showed that administration 24 h prior to irradiation was optimal. Ginseng extract or one of its three fractions was dose adjusted and injected intraperitoneally into mice that 24 h later were irradiated, whole body, with doses ranging from 7 to 11 Gy. The LD50 in 30 days was calculated using Probit analysis. The results indicated that the water soluble extract of whole ginseng gave the best protection against gamma radiation. The isolated protein and carbohydrate fractions gave less protection, while the saponin fraction did not protect.

Risk factors associated with human cystic echinococcosis in Florida, Uruguay: results of a mass screening study using ultrasound and serology.

Sonographic evidence of asymptomatic Echinococcus granulosus lesions in the liver was found in 156 of 9,515 persons in the Department of Florida, Uruguay. The sensitivity of ELISA and latex agglutination serology compared with ultrasound was 47.6% and 28.1%, respectively, and specificity was > 85%. There was a significant positive association between positive sonography and a personal history of previous but treated Echinococcus infection while those that were seropositive but ultrasound-negative were significantly more likely to have a personal history of infection or a history of infection in their family. Prevalence of infection increased significantly with age. There was no correlation between echinococcosis and dog ownership or home slaughter of sheep but offal disposal was important, with an increased prevalence of infection of 3.2%, 2.8%, and 3.1%, respectively, in persons feeding offal to dogs or burying or burning it compared with a prevalence of 0.8-1.5% in those using other methods of disposal. Almost half the population, when questioned, seemed to have sound knowledge about E. granulosus and described correct treatment of E. granulosus in dogs but this did not affect prevalence. There was a significant positive association between infection and the presence of a fenced fruit/vegetable garden and use of rural waters, particularly the cachimba (a small dam) and the aljibe (a cistern or tank) that collect rainwater from the ground surface and roofs, respectively.

Heritable protein variants induced by exposure to ethylnitrosourea: heritability, subcellular location, and tissue distribution.

The heritability, subcellular location, and tissue distribution of liver protein alterations found in the two-dimensional electrophoresis patterns of 4 offspring from male mice treated with N-ethyl-N-nitrosourea (ENU) were studied. Mice homozygous for each of the 4 ENU-induced protein variants were found to be viable and fertile, although the number of homozygous offspring from crosses between heterozygous carriers of one variant (ENU 2) was less than that expected for a nondetrimental trait. Two of the proteins altered by ENU-induced mutations were associated with the crude mitochondrial fraction, another was found predominantly in the microsomal fraction, and the fourth was associated with the mitochondrial, microsomal, and cytosol fractions. All of the ENU-induced mutations affected proteins that were not liver-specific; i.e., the proteins were found in other tissues in addition to the liver.

The analysis of recessive lethal mutations in mice by using two-dimensional gel electrophoresis of liver proteins.

We have used two-dimensional gel electrophoresis (2DE) coupled with computer-assisted data analysis to analyze liver-protein expression in mice known to be heterozygous carriers of recessive lethal mutations induced in In(1)1Rk or In(7)13Rk inversion stocks by exposure to either triethylene melamine or ionizing radiation. Carriers of 8 different mutations and corresponding littermate controls (average of 17 individuals in each group) were screened for liver-protein differences. Both qualitative and quantitative protein differences were detected that correlated with unique pedigrees among the mouse stocks analyzed. Such strain-specific differences demonstrated that quantitative differences (either increases or decreases) in protein abundance of greater than 25% can be readily detected by using this 2DE system. Thus the 50% reduction in expression of a protein expected in the event of a structural gene deletion is well within the level of detection. No significant quantitative decreases in protein expression that correlated with the recessive lethal mutations were detected, however.

Significance of particle size of mebendazole in the treatment of tapeworm infections.

Taenia hydatigena and Echinococcus granulosus in dogs have been used as a model to evaluate the response of these parasites to increasing doses of mebendazole in different dose forms (powder, micronized powder and tablets of micronized powder). Overall the micronized powder proved to be the most active, the tablets less so and the normal powder least active. Statistical confirmation was obtained that the use of this drug in different dose forms changed the rate of the response, suggesting that in the treatment of human hydatid disease caused by Echinococcus spp and cysticercosis caused by Taenia solium, the dosage form may be a vital factor in the drug's efficacy.

The survival in sheep and infectivity to dogs of Taenia hydatigena and T. ovis in sheep.

Lambs were reared from birth to 3 months of age on pasture contaminated with the eggs of either Taenia hydatigena or Taenia ovis. They were necropsied at 3, 6, 9 or 12 months. Almost all larvae that were viable at 3 months continued to survive throughout the experiment. Larvae of T. hydatigena were infective to dogs at 3 months, whereas those of T. ovis required more than 3 months to reach the infective stage.

Control of Echinococcus granulosus in Cyprus and comparison with other island models.

A control programme directed against Echinococcus granulosus was introduced into the Republic of Cyprus in 1971. In 1974, this was restricted to the areas controlled by the government of the island. The force used in the 'attack' phase included a major dog control programme. This and other measures led to the rapid 'disappearance' of the parasite and control was terminated in 1985. Subsequent studies during 1993-1996 revealed that the parasite was present in 82 (20%) villages in either dogs or food animals or both and control was re-introduced in the 'consolidation' phase with emphasis on the surveillance of E. granulosus in intermediate hosts, animal movement control and treatment of dogs in 'infected' villages. During these surveys, a major difficulty was found to be in determining whether transmission was autocthonus or introduced by animals from the areas not controlled by the government. Evidence was found to suggest the former was the most likely for the infected animals which had been born and reared far from the dividing line in the government-controlled area. A major contribution of this and other island programmes, such as those in Iceland, New Zealand, Tasmania (Australia) and the Falkland Islands, to an understanding of control planning has been the demonstration that it cannot readily be determined when eradication has been achieved and that once the costly 'attack' phase has been converted into the much more cost-effective 'consolidation' phase this must be maintained indefinitely in that phase if there is cross-border transmission as is likely to be the case in all Continental control programmes.

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of anthelmintics for dogs and cats.

Guidelines have been designed to assist in the planning, operation and interpretation of studies for the assessment of the efficacy of drugs against helminth parasites of dogs and cats. The advantages, disadvantages and application of critical and controlled tests are presented. Information is also provided on the selection of animals, housing, feeding, dose-titration, confirmatory and clinical trials, record keeping and necropsy procedures. These guidelines should assist both investigators and registration authorities involved in the evaluation of anthelmintics to employ comparable and standard procedures and will have the added benefit of minimising the numbers of animals needed for such tests.

Control of Echinococcus granulosus in Uruguay: evaluation of different treatment intervals for dogs.

This study attempted control of transmission of Echinococcus granulosus from dogs to sheep in different areas in the Department of Florida, Uruguay, by treating dogs with praziquantel at intervals of 6, 12 and 16 weeks. The 6-week interval was based on the prepatent period of infection with E. granulosus, the 12- and 16-week intervals were based on the rate of reinfection with tapeworms in dogs in the area. Dogs had become reinfected with E. granulosus between 2 and 4 months after treatment, whereas they became reinfected with the Taenia spp. tapeworms within 2 months of treatment. One year after the start of treatments sentinel lambs were born and grazed the farms in the three treatment areas. Approximately, 15 months later when the sentinel lambs were killed and examined for parasites the six weekly treatments had stopped the transmission of E. granulosus to the sentinel lambs. Treatment of dogs at 12- and 16-week intervals failed to stop transmission of E. granulosus but both the numbers of farms and the numbers of sheep infected with E. granulosus were lower where dogs received 12 weekly treatments compared with dogs receiving 16 weekly treatments and a fourth area where dogs had received no treatments (chi(2)P=0.002). Lambs continued to become infected with the Taenia spp. tapeworms in all the areas. Control was complicated by large changes in the dog population. From a starting population of 1164 dogs in the three treated areas, 832 new dogs, most of these adult hunting dogs, entered the population and 793 dogs were lost from the population.

Factors regulating tapeworm populations: estimations of the build-up and dispersion patterns of eggs after the introduction of dogs infected with Taenia hydatigena.

The build-up of the eggs of Taenia hydatigena was measured on a pasture following the introduction of infected dogs. Susceptible lambs grazed on the pasture beforehand showed that only small numbers of randomly dispersed infective eggs were present. Two dogs each with four patent T hydatigena were chained to kennels placed in the middle of the pasture. The build-up of eggs was determined by grazing further tapeworm-free lambs there for 10-day periods. High cyst counts were obtained in some of these lambs grazed within 10 days of the introduction of the infected dogs. The mean cyst counts remained at a similar level during the 85-day investigation. This indicated that at least part of the pasture became virtually saturated with eggs shortly after the dogs were introduced and remained so while the dogs were there. Some animal management practices that favour this 'epidemic' pattern and lead to a 'cysticercosis storm' are discussed.