TP53 mutation profile in chronic lymphocytic leukemia: evidence for a disease specific profile from a comprehensive analysis of 268 mutations.

The TP53 mutation profile in chronic lymphocytic leukemia (CLL) and the correlation of TP53 mutations with allele status or associated molecular genetics are currently unknown. We performed a large mutation analysis of TP53 at four centers and characterized the pattern of TP53 mutations in CLL. We report on 268 mutations in 254 patients with CLL. Missense mutations appeared in 74% of cases compared with deletions and insertions (20%), nonsense (4%) and splice site (2%) mutations. The majority (243 of 268) of mutations were located in the DNA-binding domain. Transitions were found in 131 of 268 mutations, with only 41 occurring at methylated CpG sites (15%), suggesting that transitions at CpGs are uncommon. The codons most frequently mutated were at positions 175, 179, 248 and 273; in addition, we detected a common 2-nt deletion in the codon 209. Most mutations (199 of 259) were accompanied by deletion of the other allele (17p-). Interestingly, trisomy 12 (without 17p-) was only found in one of 60 cases with TP53 mutation (without 17p-) compared with 60 of 16 in the cohort without mutation (P=0.006). The mutational profile was not different in the cohorts with and without previous therapy, suggesting that the mechanism underlying the development of mutations may be similar, independent of treatment.

Ectopic (subcutaneous) Paragonimus miyazakii infection in a dog.

Ectopic infection with Paragonimus miyazakii was determined to be the cause of a subcutaneous inguinal mass in a 15-month-old, male, boar-hunting dog. On histologic examination, the mass comprised granulomatous panniculitis, intralesional adult trematodes and eggs, and lymphadenitis. Extrapulmonary paragonimosis in animals is rare. This appears to be the first report in a dog of ectopic P. miyazakii infection with mature trematodes and eggs that involved the inguinofemoral lymphocenter and surrounding subcutis.

Identification of the human paragonimiasis causative agent in Lao People's Democratic Republic.

To assess the species of human paragonimiasis in Lao People's Democratic Republic, 6 ovum samples from 6 native confirmed paragonimiasis patients were examined with polymerase chain reaction (PCR) amplifying the internal transcribed spacer 2 (ITS2). The PCR products were sequenced, and a homology search was performed using the GenBank. All 6 sequences were identical with Paragonimus heterotremus ITS2. Our work suggests that P. heterotremus may be the main etiological agent of human paragonimiasis in this locality.

Paragonimiasis and its intermediate hosts in a transmission focus in Lao People's Democratic Republic.

Human cases of paragonimiasis have been reported in Lao People's Democratic Republic from several provinces, however, detailed descriptions of a focus of transmission including parasites species and intermediate hosts are lacking. Field investigations were carried out between February and July 2004 in three neighboring villages of the province of Vientiane. Persons with chronic cough (more than three weeks) were identified during a door-to-door survey and three sputum samples were examined for Paragonimus spp. and tuberculosis on two consecutive days. Potential intermediate snail and crustacean hosts were sampled among the populations' water-contact sites and examined for metacercarial infections or cercarial shedding. Four dogs were infected with metacercaria and the species of the subsequently retrieved adult parasites were identified. 12.7% (15 cases) of all chronic cough patients (0.6% of the total population) suffered from paragonimiasis. Samples of five species of freshwater snails, two species of crabs and one species of prawn were collected from the Nam Sêt River. None of the snails were infected. Metacercariae of P. harinasutai, P. bangkokensis and P. heterotremus were found in Potamon lipkei crabs while an infection with P. westermani was observed in a Chulathelphusa brandti crab. No paragonimid metacercaria was found in the Macrobrachium dienbienphuense prawns Dang and Nguyen 1972. This study demonstrates the importance of Paragonimus spp. as an etiological agent in patients with chronic cough and suggests that similar transmission might be widespread in Lao People's Democratic Republic. We document herein the transmission of several Paragonimus species indicating that paragonimiasis is an active zoonosis in Lao People's Democratic Republic.

Chromosomal differentiation of the Schistosoma japonicum complex.

The C-banding pattern, location of telomere sequence and chiasma frequency of four species of the Schistosoma japonicum complex were compared with those of two African species, Schistosoma mansoni and Schistosoma haematobium. In the six species, C-banding patterns of seven autosomes and the two sex chromosomes (Z and W) showed relatively species-specific and geographical (Asian and African) differences. Particularly, a plausible pathway of alteration of chromosome 2 revealed a direction from the A-chromosome to the M- chromosome in terms of rearrangements of pericentric inversion and elimination of constitutive heterochromatin (AM inversion). This chromosome change suggested hypothetically that the S. japonicum complex is the original type, and the African species represents the derived type. Moreover, the mosaic construct of the Asian and African types in Schistosoma sinensium chromosomes prompted us to propose that the species might have been formed by hybrid speciation of the genomes of Asian and African species. Localisation of telomeric repeats enabled Asian and African schistosomes to be distinguished clearly by simple terminal location and by terminal and interstitial locations, respectively. Change of chiasma frequency in the S. japonicum complex might be caused by the reduction of interstitial chiasmate (Xi) in the larger chromosomes, 1 and Z (or W), and the change seems to have progressed to Japan from South East Asia. These data enabled us to predict a tentative evolutionary pathway of schistosomes at the cytogenetic level.

Cercarial density in the river of an endemic area of schistosomiasis haematobia in Kenya.

The cercarial density in natural water and number of infected Bulinus globosus were monitored over a one-year period to identify the transmission foci in an endemic area of schistosomiasis haematobia in Kenya. Overall prevalence and intensity of infection of the study community were 59.2% and 10.9 eggs/10 ml of urine. Cercariometry was carried out on 456 occasions at 20 study sites while snail sampling was done on 465 occasions at the same sites over a one-year period. Cercariometry was exclusively done at flowing water habitats. The results showed the focality and seasonality of transmission. Cercariae were detected on 44 occasions at 11 sites. The detections were made on seven occasions at two study sites, six occasions at one site, four occasions at four sites, three occasions at one site, two occasions at two sites, and one occasion at one site. Densities of 1-4 cercariae/100 liters of water were found on 31 occasions. Five to nine cercariae/100 liters of water were found on seven occasions, 10-19 cercariae/100 liters of water were found on two occasions, and high cercarial densities greater than 20 cercariae/100 liters of water were found on four occasions. The highest count was 52 cercariae/100 liters of water. The presence of cercariae in natural water was shown to depend on the water temperature, but the intensity and duration of sunlight did not affect the presence of cercariae in water. The monthly variability of cercarial density was proportional to the number of infected snails. Cercarial density was highest in March and April, in the middle of the rainy season, whereas no cercariae were detected in cool dry season. The snail population peaked late in March, the beginning of the long rainy season, remained high for two months, and decreased rapidly late in May when heavy rain occurred. The overall infection rate of snails was 7.3% and the majority of infected snails were collected from March to May. There was no definite correlation between the presence or absence of cercariae and infected snails. Cercariae were frequently found where infected snails were absent and cercariae were sometimes absent where infected snails were present. Cercariometry and snail sampling remain quite complementary in identifying the transmission foci of schistosomiasis.

In vivo and in vitro cellular response to Schistosoma japonicum eggs in hosts with differing susceptibilities.

A comparative study of the cellular response to Schistosoma japonicum eggs was conducted in order to explore its significance, using hosts with differing susceptibilities to the parasite. In experimentally induced, synchronized hepatic granuloma formation, animal species formed each characteristic feature of the granulomas, and the magnitude of tissue reaction was significantly larger in highly susceptible hosts, such as mice and hamsters, while less susceptible hosts, such as rats and quails, formed smaller granulomas. Confluent neutrophils were seen within the tissue lesions for mice and hamsters, while rats and quails showed obviously scanty neutrophils. Guinea pigs failed to develop any granulomas. When splenic cells and bone marrow cells were used for in vitro granuloma formation, bone marrow cells showed markedly higher reactions than splenic cells from naive or sensitized animals and the reactivities of bone marrow cells from susceptible hosts, mice and hamsters, were clearly higher than those from rats, indicating similar results to those of in vivo granuloma formation. This study indicates that the in vivo and in vitro cellular response to S. japonicum eggs varies greatly according to the host's susceptibility, independent of whether the host is a naive or sensitized animal. Our results seem to support the concept that the parasites exploit the host immune system in order to complete their life-span.

Tetraploids of the lung fluke Paragonimus westermani found in China.

Two groups of Paragonimus westermani (Tematoda: Platyhelminthes) exist in nature: diploids and triploids. Generally, these two groups live allopatrically, but in Kuandian, Liaoning Province, in the Republic of China, they live sympatrically. In our Chinese experiment on Paragonimus we used metacercariae of P. westermani, which we collected in Kuandian, Xigutai, and performed a cytological analysis. The results were as follows: (1) the P. westermani in Xigutai lived sympatrically as diploids and triploids; (2) all of the small metacercariae were diploids; (3) the large metacercariae were in large proportion triploids; (4) we found one tetraploid specimen in both the medium and the large metacercariae--this was the first time tetraploid lung flukes were discovered; (5) the somatic chromosomes of the tetraploids were different in numbers (4n = 44), but we could not find any difference in the karyotype of haploid sets and that of the diploids and the triploids; (6) unlike the triploids, during their meiosis the tetraploids produced a chromosome pairing, and we found a tendency of the large chromosomes to become quadrivalent; and (7) also unlike the triploids, a great number of spermatids were found in the tetraploid testes. Because of these findings, we can consider tetraploids to be autotetraploids, and these are probably produced by the fertilization of diploids and triploids. We also think that the gametes of tetraploids have a fertilization capability.

[Studies on the Nepalese crude drugs. XVII. On the naphthalene related compounds from the root bark of Oroxylum indicum VENT].

Nine new naphthalene related compounds (I, IV, V, VII-XII) together with four known compounds (II, III, VI, XIII) were isolated from the root bark of Oroxylum indicum Vent. (Bignoniaceae), one of the Nepalese crude drugs. Their structures were determined based on chemical and physiocochemical evidence.

Reciprocal translocation between the long arms of chromosomes four and six of Paragonimus westermani from the Philippines.

C-banding patterns have been observed in 14 specimens of Paragonimus westermani from Jaro, Leyte, the Philippines. One of them showed mutations of 2 chromosomes in spreads. Comparative analysis of C-banding patterns between the individual and standard type clarified that the mutant resulted from a reciprocal translocation between the long arms of chromosomes 4 and 6.

Electrophoretic analysis of a natural population of the Thai Paragonimus heterotremus and its genetic relationship to the three Japanese species P. miyazaki, P. ohirai and P. westermani.

A total of 18 enzymes (encoded by 20 loci) from Paragonimus heterotremus collected at a local area in Saraburi, Thailand, were electrophoretically compared with those from the three Japanese species P. ohirai, P. miyazaki and P. westermani using horizontal starch-gel electrophoresis. Relative genetic distances between these species were quantified. The Thai P. heterotremus was found to be most closely related to P. miyazaki and rather distantly related to P. westermani. On the other hand, among the three Japanese species, the closest relationship was observed between P. miyazaki and P. ohirai.

Surface ultrastructure of newly excysted metacercariae of Paragonimus heterotremus.

The newly excysted metacercariae of P. heterotremus were examined by scanning electron microscopy (SEM). Large dome-shaped papillae, small ones with a pit, and small ones with a smooth surface were observed around the suckers. The number of the small dome-shaped papillae with a pit was about 30 around the oral sucker and that of the small ones with a smooth surface varied from 9 to 13 around the ventral sucker. The present report is the first record of excysted metacercariae of P. heterotremus examined by SEM.

Genetic variability and differentiation of natural populations in three Japanese lung flukes, Paragonimus ohirai, Paragonimus iloktsuenensis and Paragonimus sadoensis (Digenea: Troglotrematidae).

The genetic differences among 3 closely related species, Paragonimus ohirai, Paragonimus iloktsuenensis and Paragonimus sadoensis, were assessed by electrophoretic analysis of allozymes. Fifteen enzymes were analyzed using starch gel electrophoresis. Seven of 18 loci were polymorphic. The most variable population (POs) was polymorphic at 6 of the loci (P = 0.333) with a heterozygosity H of 0.054. Genetic distances between P. ohirai and P. iloktsuenensis, between P. ohirai and P. sadoensis, and between P. iloktsuenensis and P. sadoensis were, on the average, 0.0375, 0.0809 and 0.0567, respectively; values which are well within the usual range for conspecific populations. An especially low estimate of genetic distance was obtained between sympatric populations of P. ohirai and P. iloktsuenensis (0.00059) in Sendai, indicating that gene flow occurred between the 2 species.

Evidence for metacercarial polymorphism in lung flukes, Paragonimus ohirai and Paragonimus iloktsuenensis.

Cross breeding experiments between 2 species of lung fluke, Paragonimus ohirai and Paragonimus iloktsuenensis, were carried out using metacercarial characteristics as distinguishing markers. All metacercariae of F1 obtained were identical to those of P. ohirai. In the F2 and BF1 of F1 X P. iloktsuenensis, both the P. ohirai and P. iloktsuenensis types of metacercariae appeared. In the BF1 of F1 X P. ohirai, however, only the P. ohirai type metacercariae were produced. No intermediate type between the 2 species appeared. The results obtained demonstrate that the differences in the metacercariae, which were previously regarded as the most important characteristic for specific discrimination between these 2 flukes, are only a hereditary phenomenon within a single species. The metacercarial form, number of cyst layers, and body size seem to be controlled by a couple of alleles or very closely linked genes following simple Mendelian inheritance. Furthermore, we confirmed that reproduction in the lung flukes depends on cross-fertilization.

Paragonimus ohirai: genetic control of tetrazolium oxidase isozymes.

The Japanese lung fluke, Paragonimus ohirai, has three electrophoretic variants: F, FS, and S of tetrazolium oxidase (EC 1.15.1.1). Variant flukes were crossed in the laboratory. In both crosses, S X S and F X F, parental phenotypes appeared in all respective F1 progeny. In a cross of F X S, all F1 individuals derived from each parent showed the same phenotype (FS) indicating a heterozygote. On the other hand, from the cross of FS X S, 13 of FS and 11 S were observed from a parent (FS) while 2 FS and 1 S were recovered in three clones from the other parent (S). In the case of a cross between FS X F, a parent (F) produced 9 FS and 18 F clones in the offspring, numbers not significantly different from the expected values of Mendelian inheritance at the 0.01 level. The breeding data indicate that the tetrazolium oxidase isozymes of P. ohirai are controlled by two alleles, ToF and ToS, at a single locus.

Electrophoretic studies on enzymes of diploid and triploid Paragonimus westermani.

An enzyme analysis of diploid and triploid Paragonimus westermani was conducted using starch gel electrophoresis. In total, 16 enzymes, probably encoded by 18 loci, were studied for 3 populations of the diploid form sampled from 2 localities, and 4 populations of the triploid form from 4 localities. Comparison of the enzymes of the triploid and the diploid digeneans showed 5 different patterns: diaphorase (EC 1.6.2.2), glutamic-oxaloacetic transaminase (EC 2.6.1.1), hexokinase (EC 2.7.1.1), leucylglycylglycine aminopeptidase (EC 3.4.1.3), and phosphoglucomutase (EC 2.7.5.1). On the basis of the numbers of bands and their patterns, all individuals of the triploid are probably heterozygous at each of these 5 loci and homozygous at the remaining 13 loci. The occurrence of fixed heterozygotes found in triploid populations cannot be easily explained by only a single mutation. It is suggested that the variability may have been introduced by hybridization with a different sub-species or a closely related species and may, thus, have been maintained since the time of the origin of triploids.

The inheritance of enzyme variants of glutamic-oxaloacetic transaminase in Paragonimus ohirai.

Lung flukes, Paragonimus ohirai, from Kinosaki, Japan, characterized by 3 electrophoretic variants, FF, FS and SS, of glutamic-oxaloacetic transaminase (GOT: EC 2.6.1.1) were crossed in the laboratory. In the case of a cross between FS and SS, a parent, FS, produced 9 SS and 16 FS clones in the offspring, numbers not significantly different from that expected (1:1) from Mendelian inheritance. From the other cross FS X FF, 20 clones originating from the respective 20 miracidia were obtained from a parent FS. The phenotype segregation ratio was 6 FF and 14 FS, which is not significantly different from Mendelian inheritance expectation. Only 3 clones were obtained from the other parent FF, but these segregated in a ratio of 2 FS: 1 FF. These breeding data indicate that the GOT isozymes of P. ohirai are controlled by 2 codominant alleles, GotF and GotS at a single locus whose products aggregate randomly, forming a dimer.