Morphological and cytochemical characterization of the peripheral blood cells of farmed streaked prochilod Prochilodus lineatus (Characiformes, Prochilodontidae) / [Caracterização morfológica e citoquímica das células do sangue periférico do curimatã Prochilodus lineatus (Characiformes, Prochilodontidae) em cultivo]

Morphological and cytochemical studies of peripheral blood cells of fish have improved the understanding of their functions and cell types. The present study performed the Morphological and cytochemical analysis of the peripheral blood of Prochilodus lineatus, Characiform native to South America, which has been gaining space in local aquaculture and as a species introduced in Asia. Our analysis provided information on the morphological and cytochemical characteristics of the leukocytes, for the formulation of hypothesis about their role in the immune system of the species. It was found that Prochilodus lineatus has morphological and cytochemical features in common with other fish species, mainly of the Characiformes order. However, we detected the presence of heterophils and PAS positive granulocytes simultaneously with neutrophils. We also found that heterophils and PAS positive granulocytes are very similar, both morphologically and cytochemically.(AU)

O estudo da morfologia e da citoquímica das células do sangue periférico dos peixes tem sido eficaz para o entendimento de suas funções e dos tipos celulares. Este estudo realizou a análise morfológica e citoquímica do sangue periférico de Prochilodus lineatus, caracídeo nativo da América do Sul que vem ganhando espaço na aquicultura local e como espécie introduzida na Ásia. Essa análise forneceu informações sobre a morfologia e as características citoquímicas dos leucócitos, visando a hipóteses sobre suas funções. Verificou-se que estas são semelhantes em vários aspectos a outras espécies, principalmente da ordem Characiformes. No entanto, neste estudo detectou-se a presença dos heterofilos e da célula granulocítica especial, simultaneamente à presença dos neutrófilos. Ainda, foi verificado que os heterofilos e a célula granulocítica especial são muito semelhantes morfológica e citoquimicamente.(AU)

Nonlinear regression analysis of length growth in cultured rainbow trout / [Análise não linear do crescimento em comprimento de truta-arco-íris de aquicultura ]

Length growth as a function of time has a non-linear relationship, so nonlinear equations are recommended to represent this kind of curve. We used six nonlinear models to calculate the length gain of rainbow trout (Oncorhynchus mykiss) during the final grow-out phase of 98 days under three different feed types in triplicate groups. We fitted the von Bertalanffy, Gompertz, Logistic, Brody, Power Function, and Exponential equations to individual length-at-age data of 900 fish. Equations were fitted to the data based on the least square method using the Marquardt iterative algorithm. Accuracy of the fitted models was evaluated using a model performance metrics combining mean squared residuals (MSR), mean absolute error (MAE) and Akaike's Information Criterion corrected for small sample sizes (AICc). All models converged in all cases tested. Evaluation criteria for the Logistic model indicated the best overall fit (0.67 of combined metric MSR, MAE and AICc) under all different feeding types, followed by the Exponential model (0.185), and the von Bertalanffy and Brody model (0.074, respectively). Additionally, ∆AICc results identify the Logistic and Gompertz models as being substantially supported by the data in 100% of cases. The logistic model can be suggested for length growth prediction in aquaculture of rainbow trout.(AU)

O crescimento em comprimento em função do tempo tem uma relação não linear; por isso, funções não lineares são recomendáveis para descrever essa relação. Seis modelos não lineares foram usados para calcular o ganho em comprimento de truta-arco-íris (Oncorhynchus mykiss) durante 98 dias, na fase final da engorda, submetidas a três dietas diferentes em grupos triplicados. Foram ajustadas as equações de von Bertalanffy, Gompertz, logístico, Brody, função potencial e exponencial a dados individuais de comprimento-idade de 900 peixes. O ajuste foi feito pelo método dos mínimos quadrados, usando-se o algoritmo iterativo de Marquardt. A precisão do ajuste foi avaliada pelo uso de critérios combinados de ajuste: quadrado médio do resíduo (QMR), erro médio absoluto (EMA) e o critério de informação de Akaike corrigido para tamanhos amostrais pequenos (AICc). Todos os modelos atingiram a convergência para cada caso avaliado. Os critérios de avaliação do modelo logístico indicaram o melhor ajuste geral (0,67 vez dos critérios combinados MSR, MAE e AICc) para cada grupo de peixe avaliado, seguido pelo modelo exponencial (0,185) e os modelos von Bertalanffy e Brody, com 0,074, respectivamente. Similarmente, os resultados de ΔAICc identificaram-se ao modelo logístico e ao de Gompertz, com grande suporte das informações em 100% dos casos. Por fim, o modelo logístico pode ser sugerido na predição do crescimento em comprimento de truta-arco-íris cultivada.(AU)

The effect of environmental colour on the growth, metabolism, physiology and skin pigmentation of the carnivorous freshwater catfish Lophiosilurus alexandri.

The growth, physiology and skin pigmentation of pacamã Lophiosilurus alexandri juveniles were evaluated in an experiment using different tank colours (white, yellow, green, blue, brown and black) over an 80 day period. The tank colours did not cause significant differences to final body mass, total length, survival rate, carcass composition (moisture, crude protein, ash, ether extract, calcium, phosphorus, energy), or to plasma protein, triglyceride and cholesterol values. Haematocrit values, however, were highest for fish kept in white tanks (ANOVA P < 0·05), while the greatest haemoglobin levels were recorded for fish kept in blue and brown tanks (P < 0·01). The concentrations of cortisol (P < 0·001) and glucose (P < 0·01) were the most in fish in the black tanks. Tank colour affected skin pigmentation significantly, with fish in white tanks having the highest values of L* (brightness) and the lowest values in blue and black tanks. L*, however, decreased in all treatments throughout the experiment. C*ab increased significantly over the course of the experiment in fish kept in white tanks. Similar increases of C*ab were recorded in the other treatments but to a lesser extent. The use of black tanks during the cultivation of L. alexandri caused stress and should be avoided. Cultivation in white and yellow tanks produced individuals with a pale skin colour, while cultivation in blue and black tanks resulted in juveniles with a darker and more pigmented skin.

Serum Wisteria floribunda agglutinin-positive Mac-2-binding protein for patients with chronic hepatitis B and C: a comparative study.

We compared Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA+ -M2BP) levels between patients with chronic hepatitis B (n=249) and chronic hepatitis C (n=386) based on the degree of liver fibrosis. We examined WFA+ -M2BP levels in patients with F4 (cirrhosis), F3 or more (advanced fibrosis) and F2 or more (significant fibrosis) in the two groups. We further examined the relationship between five fibrosis markers and the degree of fibrosis. The WFA+ -M2BP values ranged from 0.25 cut-off index (COI) to 12.9 COI in patients with hepatitis B and 0.34-20.0 COI in patients with hepatitis C (P<.0001). The median WFA+ -M2BP values in F4 in the two groups were 2.83 COI in patients with hepatitis B and 5.03 COI in patients with hepatitis C (P=.0046). The median WFA+ -M2BP values in F3 or more in the two groups were 1.79 COI in patients with hepatitis B and 3.79 COI in patients with hepatitis C (P<.0001). The median WFA+ -M2BP values in F2 or more in the two groups were 1.49 COI in the hepatitis B cohort and 3.19 COI in the hepatitis C group (P<.0001). Among five liver fibrosis markers, WFA+ -M2BP had the highest correlation coefficient (rs =.629) in terms of correlation with the degree of fibrosis in the patients with hepatitis C and had the second highest rs value (.415) in the hepatitis B group. Although WFA+ -M2BP could be a useful indicator of liver fibrosis, WFA+ -M2BP levels in the two groups significantly differed even in the same degree of fibrosis. Individual cut-off values in each aetiology for the degree of fibrosis should be determined.

Toxicidade aguda ao sal comum e larvicultura intensiva do jundiá Rhamdia quelen em água salobra / Acute toxicity of common salt and intensive larviculture of silver catfish Rhamdia quelen in brackish water

A tolerância de peixes de água doce à salinidade e os níveis adequados de náuplios de Artemia na alimentação durante a larvicultura são de extrema importância para a padronização dos manejos em ambientes de criação intensiva. Dessa forma, o objetivo do trabalho foi estimar a salinidade letal (SL50) para larvas de jundiá Rhamdia quelen e determinar o efeito da salinidade e da concentração de presas vivas na larvicultura intensiva. No primeiro ensaio, larvas ao final do período lecitotrófico (1,1±0,8mg) foram submetidas às salinidades de 0, 2, 4, 6, 8, 10, 15 e 20g de sal/L por um período de 96h. No segundo experimento, as larvas de jundiá, no início da alimentação exógena (1,2±0,3mg), foram submetidas a três salinidades (água doce 0, 2 e 4g de sal/L) e três concentrações de presas vivas (início: 300, 500, 700 náuplios de Artemia/larvas/dia, sendo esse montante aumentado a cada cinco dias). O experimento foi realizado em delineamento inteiramente ao acaso, em esquema fatorial 3x3, por um período de 15 dias. No experimento 1, as larvas de jundiá submetidas às salinidades de 10, 15 e 20g de sal/L morreram após 12, duas e uma hora de exposição, respectivamente. As SL50 de 72 e 96h foram estimadas em 9,93 e 4,95g de sal/L, respectivamente. No final do teste de toxicidade, não houve diferença na sobrevivência entre as salinidades de 0, 2 e 4g de sal/L. No experimento 2, não foi observado efeito da interação entre salinidade e concentração de presas para o peso e o comprimento. Quanto maior a quantidade de presas, maior o crescimento das larvas. A sobrevivência apresentou interação entre os fatores. O aumento da salinidade proporcionou uma diminuição da sobrevivência, independentemente da concentração de presas. Dessa forma, conclui-se que a SL50 diminuiu com o aumento do tempo de exposição à água salinizada e que a larvicultura da espécie pode ser realizada em salinidades de até 2g de sal/L, com concentração de presas vivas diária inicial de 700 náuplios de Artemia/larva.(AU)

The tolerance of freshwater fish to salinity and the adequate levels of Artemia nauplii in the feeding regime during larviculture are of extreme importance to the standardization of management practices in intensive production environments. Therefore, the aim of this study was to estimate the lethal salinity (LS50) of the silver catfish Rhamdia quelen larvae and determine the effect of salinity and the concentrations of live prey in intensive larviculture of this species. In the first trial, larvae at the end of the lecithotrophic period (1.1±0.8mg) were subjected to salinities of 0, 2, 4, 6, 8, 10, 15 and 20g of salt/L for a period of 96h. In the second experiment, the catfish larvae starting the exogenous feed (1.2±0.3mg) were subjected to three salinities (freshwater 0, 2 and 4g of salt/L) and three concentrations of live prey (starting at: 300, 500, 700 Artemia nauplii/larvae/day, this amount being increased every five days). The experiment was conducted in a completely randomized design in a 3x3 factorial scheme, for a period of 15 days. In experiment 1, the catfish larvae subjected to the salinities of 10, 15 and 20g of salt/L died after 12, 2 and 1h of exposure, respectively. The LS50 at 72 and 96 h were estimated at 9.93 and 4.95g of salt/L, respectively. At the end of the toxicity test, there was no difference in the survival among the salinities of 0, 2 and 4g of salt/L. In experiment 2 no significant interaction was observed between salinity and the concentration of prey for weight and length. The increased quantity of prey increased the growth of the larvae. The rise in the salinity correlated to a decrease in survival, regardless of the prey concentration. Thus, it is concluded that the LS50 decreased with the increase in time of exposure to saltwater, and that the larviculture of this specie can be conducted in salinities of up to 2g salt/L, with a daily prey concentration starting at 700 Artemia/larvae.(AU)

Performance and nutrient utilization in South American juvenile catfish Pseudoplatystoma spp. weighting 89 - 170g, fed at different energy and protein levels / Desempenho e utilização de nutrientes em juvenis de surubim Pseudoplatystoma spp. alimentados com diferentes níveis de energia e proteína

Most studies in nutrition for the South American catfish (surubim) were limited to the initial phase of development. However, it is clear that performance and nutrient utilization can change during the life stages of a fish. Therefore, this study aimed to evaluate the performance and nutrient utilization in juveniles of surubim fed diets varying in protein and energy levels. Two experiments were performed to test different levels of energy and protein in formulated diets. In the first experiment, surubim juveniles (89.2±4.8g) were fed five diets containing different levels of energy (18.0, 18.8, 19.6, 20.5, 21.3 MJ/kg). In the second experiment, juveniles (170.03±3.35g) were fed five diets containing different levels of protein (360, 400, 440, 480 and 520g/kg). The most favorable energy level for weight gain was 20.3 MJ/kg. The increasing energy levels provided a rise in fat and decrease in protein whole-body composition. The protein amount was between 360 to 400g/kg (383g/kg), which was adequate for performance and nutrient assimilation in surubim juveniles.

A maior parte dos estudos a respeito dos aspectos nutricionais do surubim está limitada às primeiras fases de desenvolvimento. Entretanto, é claro que o desempenho e a utilização dos nutrientes podem mudar durante os diferentes estágios de desenvolvimento destes animais. Assim sendo, este estudo teve como objetivo avaliar o desempenho e a utilização de nutrientes em juvenis de surubim alimentados com dietas contendo níveis variáveis de energia e proteína. Dois experimentos foram realizados para testar os diferentes níveis de proteína e energia. No primeiro experimento, juvenis de surubim (89,2±4,8g) foram alimentados com cinco dietas contendo níveis diferentes de energia (18.0, 18.8, 19.6, 20.5, 21.3MJ/kg). No segundo experimento, os juvenis (170,03±3,35g) foram alimentados com dietas contendo cinco níveis de proteína (360, 400, 440, 480 e 520g/kg). O melhor nível de energia para ganho de peso foi 20,3 MJ/kg. O aumento dos níveis de energia levou a um incremento nos níveis de lipídeo e diminuição da proteína corporal. Níveis de proteína entre 360 a 400g/kg foram os mais adequados para o desempenho e utilização dos nutrientes em juvenis grandes de surubim.

Study of the prediction system for clinical response to M-VAC neoadjuvant chemotherapy for bladder cancer.

Neoadjuvant chemotherapy for invasive bladder cancer, involving a regimen of M-VAC, can manage micrometastasis and improve the prognosis. However, some patients suffer from severe adverse drug reactions without any effect, and no method yet exists for predicting the response of an individual patient to chemotherapy. Our purpose in this study is to establish a method for predicting the response to the M-VAC therapy. We analyzed gene-expression profiles of biopsy materials from 40 invasive bladder cancers using a cDNA microarray consisting of 27 648 genes, after populations of cancer cells had been purified by laser-microbeam microdissection. We identified 14 predictive genes that were expressed differently between nine responder and nine non-responder tumors and devised a prediction-scoring system that clearly separated the responder group from the non-responder group. This system accurately predicted the clinical response for 19 of the 22 additional test cases. The group of patients with positive predictive scores had significantly longer survival times than that with negative scores. As real-time RT-PCR data were highly concordant with the cDNA microarray data for those 14 genes, we developed a quantitative RT-PCR-based prediction system that could be feasible for routine clinical use. Taken together, our results suggest that the sensitivity of an invasive bladder cancer to the M-VAC neoadjuvant chemotherapy can be predicted by expression patterns in this set of genes, a step toward achievement of "personalized therapy" for treatment of this disease.

Mutational screening of ACVR1 gene in Brazilian fibrodysplasia ossificans progressiva patients.

Fibrodysplasia ossificans progressiva (FOP) is a severe genetic disorder reported worldwide. A specific heterozygous mutation (c.617G> A; p.R206H) in the activin A type I receptor gene (ACVR1) is regarded as the genetic cause of FOP in all classically affected individuals worldwide. However, a few patients with FOP variants harbor distinct mutations in ACVR1. We screened a group of FOP Brazilian population for mutations in ACVR1. Of 16 patients with a classic FOP phenotype (10 males and 6 females, age range of 3-42 years), all had the classic mutation (p.R206H). One 21-year-old woman with a variant FOP phenotype had the previously reported c.983G> A mutation (p.G328E). Our study contributes to the understanding of the predominant FOP phenotype and genotype and suggests that variant FOP phenotypes are associated with specific mutations in ACVR1 gene.

Involvement of upregulation of DEPDC1 (DEP domain containing 1) in bladder carcinogenesis.

In an attempt to disclose mechanisms of bladder carcinogenesis and discover novel target molecules for development of treatment, we applied a cDNA microarray to screen genes that were significantly transactivated in bladder cancer cells. Among the upregulated genes, we here focused on a novel gene, (DEPDC1) DEP domain containing 1, whose overexpression was confirmed by northern blot and immunohistochemical analyses. Immunocytochemical staining analysis detected strong staining of endogenous DEPDC1 protein in the nucleus of bladder cancer cells. Since DEPDC1 expression was hardly detectable in any of 24 normal human tissues we examined except the testis, we considered this gene-product to be a novel cancer/testis antigen. Suppression of DEPDC1 expression with small-interfering RNA significantly inhibited growth of bladder cancer cells. Taken together, these findings suggest that DEPDC1 might play an essential role in the growth of bladder cancer cells, and would be a promising molecular-target for novel therapeutic drugs or cancer peptide-vaccine to bladder cancers.

Endoscopic submucosal dissection for early gastric cancer: results and degrees of technical difficulty as well as success.

BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) is a new method for the curative treatment of early gastrointestinal neoplasms, which was developed in order to increase the en bloc and R0 resection rate, especially for lesions larger than 20 mm in diameter. Drawbacks of ESD include the fact that it is technically a substantially more difficult procedure and that it is associated with a higher perforation rate. A retrospective study was therefore carried out to analyze cases in relation to the procedure time and resection success, and these factors were correlated with the characteristics of the lesions. PATIENTS AND METHODS: From January 2002 to November 2005, 196 lesions in 185 patients with early gastric cancer were treated using ESD in our hospital. The rates of curative en bloc resection, the incidence of perforation, and the procedure times were analyzed in relation to lesion size (small, 20 mm or less in diameter; large, over 20 mm), location (upper, middle, or lower third of the stomach) and the presence or absence of ulceration. RESULTS: The rate of curative en bloc resection was 84 % (93 % of the lesions overall were resected in one piece), with a perforation rate of 6.1 % (all perforations were managed endoscopically) and a mean procedure time of 68 min. The rate of curative en bloc resection differed significantly depending on the location of the lesion (upper vs. middle vs. lower, 74 % vs. 77 % vs. 91 %; P < 0.05), as well as on the size of the lesion (> 20 mm vs. 20 mm or less, 59 % vs. 89 %; P < 0.0001). There were also significant differences in the mean procedure times in relation to the location of the lesion (upper vs. middle vs. lower, 105 min vs. 81 min vs. 45 min; P < 0.0001) and the size of the lesion (> 20 mm vs. 20 mm or less, 124 min vs. 55 min; P < 0.0001), as well as the presence of ulceration (positive vs. negative, 97 min vs. 65 min; P < 0.05). With regard to perforation rates, significant differences were also observed in relation to the location of the lesion (upper vs. middle vs. lower, 22.6 % vs. 2.8 % vs. 3.2 %; P < 0.0005) and size of the lesion (> 20 mm vs. 20 mm or less, 16.2 % vs. 3.8 %; P < 0.005). No local recurrences of curatively resected lesions (n = 119) were observed after a follow-up period of 1 year. CONCLUSIONS: The difficulty of ESD depends on the location and size of the lesion, as well as on the presence of ulceration. We would recommend that trainees should begin by carrying out ESD on lesions with a diameter of less than 20 mm without ulceration that are located in the lower third of the stomach.

Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27.

The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1lambdaT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1 x 10(6) times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.

Targeted disruption of sti35, a stress-responsive gene in phytopathogenic fungus Fusarium oxysporum.

sti35 is one of the heat-shock genes in Fusarium oxysporum, which is a fungal pathogen for wilt disease in plants. We have isolated a genomic clone of sti35 and used it to create disruption mutations. Disruption of the sti35 coding region resulted in the loss of a 32-kDa protein present in heat-shocked cells. The disruption had no detectable effect on growth and development at various temperatures, nor on the ability to acquire thermotolerance in nutrient medium. But the sti35 disruptants showed increased thermotolerance, relative to the wild-type strain, when incubated in minimal medium after heat treatment.

Detection and cloning of the gene encoding a protein produced by nonpathogenic mutants of Fusarium oxysporum.

Treatment of Fusarium oxysporum with 5-azacytidine, a potent inhibitor of DNA methylation, induced nonpathogenic mutants. Analysis of the protein expression pattern by two-dimensional gel electrophoresis revealed a protein that is present in yeast-form cells of the mutants but absent in those of the wild-type strain. N-terminal amino acid analysis indicated that this protein is identical to a region of a polypeptide encoded by a cDNA clone, sti35, previously identified as a heat shock gene in F. oxysporum. A genomic clone for sti35 was isolated and sequence analysis revealed an intron and two heat shock elements upstream of sti35. The analysis also revealed the presence of a leader sequence composed of 27 amino acid residues, which shares a common amino acid composition with leader sequences of the proteins located in the mitochondrial matrix. Different expression patterns of sti35 in the mutants and wild-type strain were demonstrated.

Cloning and sequencing of the Pz-peptidase gene from Bacillus licheniformis N22.

Pz-peptidase is an endopeptidase that cleaves the synthetic substrate Pz-peptide (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg), which was originally developed for the assay of collagenase. The Pz-peptidase gene of Bacillus licheniformis N22 was cloned and sequenced. The gene consists of 628 amino acids with a motif for zinc-dependent metalloprotease, and shares 42% amino acid identity with the oligoendopeptidase of Lactococcus lactis. This is the first report on the gene structure of a Pz-peptidase.

Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1.

Bacillus ehimensis EAG1 (IFO15659) produced and secreted chitosanase in the presence of exogenous chitosan. The chitosanase was purified from the culture filtrate of the bacterium to apparent homogeneity in SDS-polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 31,000. A 1.9-kbp DNA fragment containing the chitosanase gene was cloned and the complete nucleotide sequence was determined. The sequence was found to contain a single open reading frame encoding a protein of 302 amino acids. The deduced amino acid sequence showed significant homology with the chitosanase from Bacillus circulans MH-K1.

Clinical diagnosis of heterozygous dystrophin gene deletions by fluorescence in situ hybridization.

Two-thirds of patients affected by Duchenne or Becker muscular dystrophy (DMD/BMD) carry large intra-genic deletions in the dystrophin gene. In males, the deletions can be efficiently detected using multiplex polymerase chain reaction (PCR) and Southern blotting. In contrast, deletion detection in carrier females is complicated by the presence of a normal gene copy on the second X-chromosome. We have analyzed the boundaries of 570 deletions and 34 duplications in the dystrophin gene identified in the São Paulo and Leiden diagnostic laboratories. The data were used to select an optimal set of cosmid probes for the detection of the most frequently deleted areas of the dystrophin gene. Six cosmids were evaluated in fluorescence in situ hybridization (FISH) experiments to assess deletions in 21 heterozygous deletion-carriers and nine controls. No discrepancy was found between the FISH analysis and the molecular data, demonstrating the accuracy of the technique for carrier detection in Duchenne and Becker muscular dystrophy.

Antibody fragments as inhibitors of Japanese radish acid phosphatase.

VH (heavy-chain variable region) and VL (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VH and identical VL domains. Initially, the VH and VL genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified VH and VL genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.

Novel point mutations in the dystrophin gene.

Duchenne (DMD) and Becker (BMD) type muscular dystrophies are allelic X-linked recessive disorders caused by mutations in the gene encoding dystrophin. About 65% of the cases are caused by deletions, while 5-10% are duplications. The remaining 30% of affected individuals may have smaller mutations (point mutations or small deletions/insertions) which cannot be identified by current diagnostic screening strategies. In order to look for pathogenic small mutations in the dystrophin gene, we have screened the 18 exons located in the hot spot region of this gene through two different single strand conformation polymorphism (SSCP) conditions. Five different pathogenic mutations were identified in 6 out of 192 DMD/BMD patients without detectable deletions: 2 nonsense, 1 bp insertion, 1 bp deletion and 1 intronic. Except for the intronic change, which alters a splice site, all the others cause a premature stop codon. In addition, 8 apparently neutral changes were identified. However, interestingly, one of them was not identified in 195 normal chromosomes, although it was previously described in a DMD patient from a different population. The possibility that this mutation may be pathogenic is discussed. Except for two neutral changes, all the others are apparently here described for the first time.

A high performance system for molecular dynamics simulation of biomolecules using a special-purpose computer.

GRAPE (GRavity PipE) processors are special purpose computers for simulation of classical particles. The performance of MD-GRAPE, one of the GRAPEs developed for molecular dynamics, was investigated. The effective speed of MD-GRAPE was equivalent to approximately 6 Gflops. The precision of MD-GRAPE was good judging from the acceptable fluctuation of the total energy. Then a software named PEACH (Program for Energetic Analysis of bioCHemical molecules) was developed for molecular dynamics of biomolecules in combination with MD-GRAPE. Molecular dynamics simulation was performed for several protein-solvent systems with different sizes. Simulation of the largest system investigated (27,000 atoms) took only 5 sec/step. Thus, the PEACH-GRAPE system is expected to be useful in accurate and reliable simulation of large biomolecules.