[Attention deficit disorder: multidisciplinary observational study over 1 year]. / Le trouble déficitaire de l'attention, avec ou sans hyperactivité (TDA/H) : une approche pluridisciplinaire longitudinale croisée de 36 enfants.

INTRODUCTION: In France, attention deficit disorder (ADHD) has traditionally met with two opposing approaches (biological and psychoanalytic). This conflict led us to conduct a multidisciplinary observational study, on a group of 36 children over a period of 1 year. METHODS: Thirty-six children with ADHD diagnostic (DSM IV), not treated by MPH were included. Initial "multi-field" evaluation (T0) consisted of: neuro-paediatric consultation (Conners questionnaire, Child Behaviour Checklist, reading and writing scores by French tests); semi-structured child psychiatric interview (DSM-IV axis I), structural hypothesis (CFTMEA), existence of narcissistic fragility, parents/child interactions; neuropsychological standardized evaluation (attention and executive functions); psychodynamic interview and projective tests (Rorscharch, CAT or TAT). A therapeutic project is proposed combining MPH and psychotherapy according to the results. A new evaluation 1 year later (T1) included a consultation and a neuropsychological evaluation. RESULTS T0: All parental questionnaires appreciating attention deficit and hyperactivity/impulsivity were significantly pathological. The neuropsychological evaluation showed usual characteristics of ADHD with individual differences. The psychiatric evaluation revealed the frequency of comorbidity in axis I (23% of children with more than two diagnoses, 57% with anxiety disorder, 23 and 3% with oppositional and conduct disorder). FOLLOW UP (T1): Thirty-one children were re-examined (20 treated by MPH and 11 not treated because of parental refusal or particular psychopathological situations). Psychoanalytical psychotherapy, proposed to 28 children, was undertaken with only 19. An improvement in scores for attention and executive tests was registered only in the treated group. DISCUSSION: The tests confirm deficits of attention and executive functions without correlation with the scores of questionnaires, underlining the need for a neuropsychological evaluation to objectify attention disorders. Projective tests refine and enrich psychiatric evaluation and showed that half of the children had borderline organization. However, structural hypotheses were heterogeneous, suggesting the need for specific therapeutic projects to be devised according to each child. The treated children were the only ones to improve attention deficit. On the other hand, the scores of anxiety are not improved by MPH, emphasizing the indications of psychotherapy if comorbidity is present. Psychotherapeutic care was carried out only among part of the population, because of parental reservations, exacerbated by differences of opinion among professionals and lack of access. CONCLUSION: This study is innovative, providing precise data on ADHD from a multidisciplinary perspective. Psychopathological comorbidity is high in this population, so the concept of ADHD cannot be limited to a cognitive point of view. These elements and doubts regarding the efficacy of behavioural therapies suggest the need for a rigorous evaluation of analytical psychotherapies independent of MPH to treat attention deficit.

[Evolution of competence in reading, spelling and comprehension levels in low socioeconomic environments and impact of cognitive and behavioral factors on outcome in two years]. / Evolution des compétences en lecture, compréhension et orthographe en environnement socioéconomique défavorisé et impact des facteurs cognitifs et comportementaux sur le devenir à deux ans.

BACKGROUND: The prevalence of poor reading skills is particularly high among children from low socioeconomic backgrounds, but no longitudinal studies have been conducted so far in France to determine whether poor reading in a socioeconomically challenged population is persistent and warrants preventive action. DESIGN: One hundred and fifty-four children were divided into three groups according to their reading skills: poor, intermediate and typical readers. They were followed over a period of 2 years. Reading levels, spelling and comprehension were assessed by standardized measurement scales in order to determine reading outcome and predictive variables. RESULTS: The reading skills in each group progressed at similar rates, but the differences between the three groups remained relatively constant over the 2 years. The gap between good and poor readers actually increased for the poorest readers. Spelling scores followed a similar pattern and remained weak. Comprehension scores followed a different pattern. Most of the initially poor readers with low comprehension scores almost caught up and reached the level of the typical readers. The best predictive variables of reading and spelling outcome were phonological awareness, rapid naming and attention deficit. The strongest predictive variables for comprehension were IQ, lexical level and attention. CONCLUSIONS: Our results confirm the relative stability of reading measurement across time in poor readers from low socioeconomic backgrounds. Their behavior are similar to the classic dyslexic population. The predictive variables are different depending on whether reading or spelling or comprehension is considered. These results provide a clear agenda for preventive literacy action in children with low socioeconomic levels (SES): phonological decoding and oral language skills in early grades, and screening and treatment of attention disorders.

[Deficits in reading acquisition in primary school: cognitive, social and behavioral factors studied in a sample of 1062 children]. / Troubles d'acquisition de la lecture en cours élémentaire : facteurs cognitifs, sociaux et comportementaux dans un échantillon de 1062 enfants.

BACKGROUND: Reading impairment is the major learning disability in children. While research on illiteracy has mainly been conducted from a sociological perspective, research on dyslexia has typically been studied from a cognitive-linguistic perspective. Studies that jointly investigate sociological, behavioral and cognitive factors in predicting reading outcome are rare and limited to English-speaking populations. The goal of the present study was to screen second grade children with reading impairment in French urban elementary schools and to pin down the factors that explain the various facets of reading failure and success. METHODS: A total of 1062 children from 20 different schools in the city of Paris participated in the study. Different aspects of reading were assessed individually for children with a suspected impairment in reading acquisition. Subsequently, 131 poor readers and 50 typically developing readers were matched for sex, age, and school. For these children, medical, cognitive, behavioral and individual socioeconomic data were obtained. Group differences were examined and multiple regression analyses were conducted to examine how much variance in reading was explained by the various variables. RESULTS: The prevalence of poor reading skills in grade 2 was highly influenced by neighborhood socioeconomic status (SES) (ranging from 3.3% in high SES to 20.5% in low SES areas). Among the SES variables, employment of the father was a significant predictor of poor reading. Among the cognitive variables, phonological awareness and rapid naming were the most significant factors, much more than verbal or nonverbal intelligence. Among the behavioral variables, attention was an important factor but not externalized symptoms. Multiple regression analyses showed that reading outcome was best predicted by phonological awareness skills and attention deficits. CONCLUSION: The majority of children with reading disability come from low SES areas. As in the English literature, the most robust predictor for reading impairment is phonological awareness, even when SES is taken into account. In addition, attention deficits seemed to aggravate reading impairments for children with weak phonological awareness skills. Successful early prevention should focus on reinforcing phonological awareness, recoding and attention skills.

Apoptotic effects on B-cell chronic lymphocytic leukemia (B-CLL) cells of heterocyclic compounds isolated from Guttiferaes.

A series of 10 heterocyclic compounds purified from Allanblackia were tested on two B cell lines, ESKOL and EHEB, and on cells from B-CLL patients. Several molecules inhibited the proliferation of both cell lines and promoted apoptosis of B-CLL cells through different mechanisms, some of them elicited a dissipation of the mitochondrial transmembrane potential, other triggered caspase-3 activation and cleavage of the inducible nitric oxide synthase. Blood mononuclear cells and B-lymphocytes from healthy donors appeared less sensitive than B-CLL cells. These results indicate that these molecules may be of interest in the development of new therapies for B-CLL.

[The rapid development of child neuropsychology]. / L'essor de la neuropsychologie de l'enfant.

The past few years have seen important progress and new developments in the field of child neuropsychology. Children can exhibit acquired or learning disorders, but in all cases such deficits take place in a developmental trajectory that requires taking into account cerebral maturation and plasticity processes. Child neuropsychology finds its specificity in the perspective of developmental neuropsychology and has close connections with health and education. This article focuses on two pathologies - childhood epilepsies and developmental dyslexia - that highlight the recent progress in this specialty and its perspectives.

[Prevalence of reading disabilities in early elementary school: impact of socioeconomic environment on reading development in 3 different educational zones]. / Prévalence des troubles d'apprentissages du langage écrit en début de scolarité : l'impact du milieu socioéconomique dans 3 zones d'éducations distinctes.

OBJECTIVE: Socioeconomic status (SES) has a known influence on academic achievement. Most studies, however, were conducted in English-speaking countries. Because recent cross-linguistic studies suggest that reading English is much harder to learn than reading other languages, an epidemiological study was conducted in French investigating the impact of socioeconomic background on early reading development. DESIGN: One thousand and twenty second-grade children (476 girls and 544 boys) from 20 different schools participated in the study. Approximately 1/3 of the children lived and were schooled in a high SES area, 1/3 in an intermediate SES area, and one final third in a very low SES area. Assessment of reading, writing and mathematical skills was conducted initially in small groups. Children with suspected learning difficulties were further tested individually. Forty-two children of equivalent age who repeated the first grade received similar individual testing. RESULTS: Average reading scores were in accordance with chronological age, without gender differences. Children from low SES schools had academic performances significantly lower than their peers. Boys exhibited superior arithmetic skills than girls. A significant reading delay was observed in 12.7% of children. The prevalence of poor reading was highly correlated with the area of schooling, varying from 3.3% in the high SES area to 24.2% in low SES area. CONCLUSION: The high rate of children from our sample with a significant delay in reading depended on general socioeconomic environment. An understanding of the origin of such differences is mandatory for defining and coordinating preventive actions and appropriate interventions.

[Study of causal factors of reading impairment in a sample of 1062 7 to 8-year-old children]. / Etude des facteurs liés aux difficultés d'apprentissage de la lecture. A partir d'un échantillon de 1062 enfants de seconde année d'école élémentaire.

OBJECTIVE: From an original large sample of 1062 7 to 8-year-old children, reading skills were assessed and found to be highly linked with socioeconomic status (SES). The purpose of the present study was to further determine underlying medical, sociocultural, cognitive and behavioural factors explaining the diversity of reading skills and the influence of SES. METHOD: Individual testing among low-SES children identified 100 poor readers, 50 typical readers and 31 children with intermediate reading scores. All 3 groups underwent a thorough assessment, including a medical evaluation, a full cognitive battery, a structured parental interview and behavioural questionnaire. Logistic regression was used to demonstrate the variables predicting reading score outcome. RESULTS: None of the medical factors studied was statistically related to reading scores. Due to the methodology buffering the impact of SES sociocultural variables, such as parental levels of education, parental occupation, as well as familial income were weak, but statistically significant predictors. The strongest variables were phonological abilities and symptoms of attention disorders. In a final regression model, phonological awareness, level of mother's education and attention explained the differences in reading skills. CONCLUSIONS: These results, which are unique in France, are similar to existing data in the literature. They support the need to conceptualize an early screening programme to detect reading difficulties and to promote an intervention based on phonological processing and decoding in low-SES environments.

Hyperforin, a new lead compound against the progression of cancer and leukemia?

Extracts of the plant St John's wort, Hyperforin perforatum L., have been used for centuries in traditional medicine, notably for the treatment of depression. One of their main lipophilic components, a natural prenylated phloroglucinol termed hyperforin (HF), has been identified as the major molecule responsible for the antidepressant effects of this plant. Within the last few years, a number of studies have demonstrated that HF displays, in addition, several other biological properties of potential pharmacological interest. They include an antibacterial capacity and inhibitory effects on inflammatory mediators. It is worth noting that HF also promotes apoptosis of various cancer cells from solid tumors and hematological malignancies, including B-cell chronic lymphocytic leukemia. In addition, HF inhibits the capacity of migration and invasion of different tumor cells, as well as exhibiting antiangiogenic effects. Altogether, these properties qualify HF as a lead structure for the development of new therapeutic molecules in the treatment of various diseases, including some malignant tumors.

Hyperforin inhibits MMP-9 secretion by B-CLL cells and microtubule formation by endothelial cells.

We previously reported that hyperforin (HF), a natural phloroglucinol purified from Saint John's wort, can induce the apoptosis of leukemic cells from patients with B-cell lymphocytic leukemia (B-CLL) ex vivo. We show here that treatment of cultured B-CLL patients' cells with HF results in a marked inhibition of their capacity to secrete matrix metalloproteinase-9, an essential component in neo-angiogenesis through degradation of the extracellular matrix process. The phloroglucinol acts by decreasing the production of the latent 92 kDa pro-enzyme. The inhibitory effect of HF is associated with a decrease in VEGF release by the leukemic cells. Moreover, HF is found to prevent the formation of microtubules by human bone marrow endothelial cells cultured on Matrigel, evidencing its capacity to inhibit vessel formation. Our results show the antiangiogenesis activity of HF and strengthen its potential interest in the therapy of B-CLL.

Pro-apoptotic properties of hyperforin in leukemic cells from patients with B-cell chronic lymphocytic leukemia.

The effects of the hyperforin (HF), a natural phloroglucinol purified from Hypericum perforatum, were investigated ex vivo on leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). HF was found to promote apoptosis of B-CLL cells, as shown by time- and dose-dependent stimulation of phosphatidylserine externalization and DNA fragmentation, by disruption of the mitochondrial transmembrane potential, caspase-3 activation and cleavage of the caspase substrate PARP-1. Moreover, HF-induced downregulation of Bcl-2 and Mcl-1, two antiapoptotic proteins that control mitochondrial permeability. HF also downregulated two proteins which are overexpressed by B-CLL patients' cells, the cell cycle inhibitor p27kip1 through caspase-dependent cleavage into a p23 form, and the nitric oxid (NO) synthase of type 2 (inducible NO synthase). This latter was accompanied by reduction in the production of NO known to be antiapoptotic in B-CLL cells. Preventing effects of the general caspase inhibitor z-VAD-fmk indicated that HF-promoted apoptosis of B-CLL cells was mostly caspase dependent. Furthermore, normal B lymphocytes purified from healthy donors appeared less sensitive to HF-induced apoptosis than B-CLL cells. These results indicate that HF may be of interest in the development of new therapies for B-CLL based on the induction of apoptosis and combination with cell cycle-dependent antitumor drugs.

[What should be done when a child cannot learn to read?]. / Que faire devant un enfant qui n'apprend pas à lire?

When a child faces difficulties in learning to read, clinicians should, first of all, examine cognitive functions. The actual reading level is checked using a test calibrated on normal reading development. They are thus able to differentiate between global learning disorders (related to mental deficiency or pervasive disorders) and specific reading impairment. Specific reading impairment might be a simple delay or developmental dyslexia. We described the first at-school training, as well as the criteria of speech therapy.

[Validation of BREV: comparison with reference battery in 173 children with learning disorders]. / BREV, une batterie d'évaluation des fonctions cognitives: validation dans les troubles des apprentissages.

INTRODUCTION: The BREV battery (Battery for rapid evaluation of cognitive functions) is a tool which can be used for the rapid neuropsychological evaluation of children aged between 4 and 9 years. OBJECTIVES: After standardization (700 unaffected children) and validation by comparison with a reference battery (202 children with epilepsy), the aim of this study was further validation in 173 children with learning disorders. POPULATION AND METHODS: The study protocol included administration of the BREV, precise neuropsychological examination and evaluation of oral and written language. Statistical analysis was used to compare the findings of the BREV with those of the reference method, and the recommendations indicated by the BREV with the final diagnoses, and to define the sensitivity and the specificity of the BREV battery. RESULTS: All the correlations between BREV tests and reference tests were significant. Recommendations after the BREV were in agreement with the conclusions of the reference evaluation in 168/172 children for language, 145/173 for the psychometric evaluation. For only 4 chidren, the results of the BREV were false negative. Diagnoses corresponded in 168/173 children for oral language, in 102/110 for written language, 166/173 for praxis disorders and 157/173 for intellectual deficit. The most predictive subtests of the BREV and sensitivity and specificity of verbal and non-verbal scores were calculated. CONCLUSION: The BREV is a reliable examination, in learning disorders, to determine the most complementary investigations both in terms of language disorders and for non-verbal or global learning disabilities.

[Heart and freezer: about one case of hypereosinophilia and coronary spasm]. / Coeur et congélateur: a propos d'un cas d'hyperéosinophilie et de spasme coronaire.

We report the case of a 41-year-old woman, non-smoker, without cardiovascular risk, hospitalised for acute ST elevation myocardial infarction. The coronarography showed no significant coronary stenosis and a methylergometrine test performed was positive. After the discovery of a moderate chronic hypereosinophilia, a review of the literature about eosinophile and coronary pathology was realised in order to make a link between hypereosinophilia and coronary spasm.

[Trisomy 21 and breast cancer: A genetic abnormality which protects against breast cancer?]. / Cancer du sein et trisomie 21 : une anomalie génétique qui protège contre le cancer du sein ?

INTRODUCTION: Trisomy 21 (T21) is the most common chromosomal abnormality and one of the main causes of intellectual disability. The tumor profile of T21 patients is characterized by the low frequency of solid tumors including breast cancer. METHODS: The objective of this work was to analyze the literature to find possible clues for the low frequency of breast cancer in T21 persons with a focus on one hand to the various risks and protective factors against breast cancer for women T21, and on the other hand to changes in the expression of different genes located on chromosome 21. RESULTS: T21 women have hormonal and societal risk factors for breast cancer: frequent nulliparity, lack of breastfeeding, physical inactivity and high body mass index. The age of menopause, earlier in T21 women, has a modest protective effect against breast cancer. The low rate of breast tumors in T21 women is probably mainly linked to the reduced life expectancy compared to the general population (risk of death before the age of onset of the majority of breast cancers) and the presence of a third chromosome 21, characterizing the disease. It might lead to the increased expression of a number of genes contributing directly or undirectly to tumor suppression, decreased tumor angiogenesis and increased cell apoptosis. Moreover, changes in the mammary stroma of persons T21 could have an inhibitory role on the development of breast tumors. CONCLUSION: The low frequency of breast cancers for T21 patients may not only be explained by hormonal and societal factors, but also by genetic mechanisms which could constitute an interesting axis of research in breast cancer.

Autocrine loop of tumor necrosis factor induced by interferon-alpha in tumor cells from hairy cell leukemia.

Interferon-alpha (IFN-alpha) is successfully used in the therapy of hairy cell leukemia (HCL). We previously reported that IFN-alpha administered to HCL patients stimulated the levels of tumor necrosis factor (TNF) receptors on hairy cells. Here, we investigated the interactions between IFN-alpha and TNF, using the HCL cell line Eskol as a model. We showed that increased levels of TNF receptors (TNFR) induced by IFN-alpha treatment were associated with stimulation of high M(r) TNFR. Low M(r) TNFR were not detected on Eskol cells or hairy cells, either before or after IFN-alpha treatment. Furthermore, the expression of TNF mRNA increased in Eskol cells treated with IFN-alpha for 1-4 h as compared to control cells. In vivo experiments showed that IFN-alpha given to HCL patients induced both TNF mRNA expression in hairy cells and secretion of active TNF protein in patients' serum. These results provide evidence that IFN-alpha may induce an autocrine loop of TNF in HCL.

Flavopiridol downregulates the expression of both the inducible NO synthase and p27(kip1) in malignant cells from B-cell chronic lymphocytic leukemia.

Flavopiridol, an inhibitor of cyclin-dependent kinases and other protein kinases, induces in vitro apoptosis of malignant cells from B-cell chronic lymphocytic leukemia (B-CLL). Previously, we reported that nitric oxide (NO), produced by an inducible NO synthase (iNOS), spontaneously expressed by the B-CLL cells, contributed to their deficiency in apoptosis. In the present work, we show that ex vivo treatment of leukemic cells from B-CLL patients with flavopiridol results in the inhibition of iNOS expression, as determined by immunofluorescence and Western blotting, and in a marked inhibition of NO production measured in situ with a specific fluorescent probe (DAF-2 DA). These effects are accompanied by membrane, mitochondrial and nuclear events of apoptosis. Flavopiridol exposure also results in the stimulation of caspase 3 activity and in caspase-dependent cleavage of p27(kip1), a negative regulator of the cell cycle, which is overexpressed in B-CLL. Thus, flavopiridol is capable of downregulating both iNOS and p27(kip1) expression in B-CLL cells. Furthermore, flavopiridol-promoted apoptosis is partly reverted by an NO donor, suggesting that inhibition of the NO pathway could participate in the apoptotic effects of flavopiridol on the leukemic cells.

Proliferative response of hairy cells to B cell growth factor (BCGF): in vivo inhibition by interferon-alpha and in vitro effects of interferon-alpha, -beta, and -gamma.

Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha therapy. In vitro, B cell growth factor (BCGF) can induce proliferation of hairy cells. We have investigated the effect of in vitro and in vivo treatments with different recombinant IFN on the capacity of hairy cells to proliferate in response to human BCGF. In vitro treatment of leukemic cells from HCL patients with recombinant IFN-alpha-2 (5/5 cases) or IFN-beta (4/5 cases) resulted in a marked inhibition of the BCGF-dependent response. This suppressive effect was obtained with IFN concentrations of 1000, 100 IU/ml, and even occasionally 10 IU/ml. In contrast, no such inhibition was observed with IFN-gamma, despite the presence of specific IFN-gamma receptors on hairy cells at densities similar to receptors for IFN-alpha/beta. The IFN-alpha-induced suppression of the proliferative response of hairy cells to BCGF was also observed in vivo in two patients within 6-12 hr after administration of single doses of IFN-alpha. When hairy cells were maintained in culture for 1 week, they recovered their capacity to be stimulated by BCGF. This reversion was also shown in vivo in hairy cells isolated 1 week after IFN administration. Since in vivo growth of hairy cells could possibly result from the autocrine secretion of BCGF, we propose that the therapeutic effect of IFN-alpha on HCL may be due in part to an inhibition of such autocrine loop.

[Specific remedial therapy in a specialist unit: evaluation of 31 children with severe, specific language or reading disorders over one academic year]. / Evaluation ouverte de l'efficacité de la prise en charge en milieu spécialisé de 31 enfants avec un trouble spécifique sévère du langage oral/écrit.

INTRODUCTION: Up to 3 percent of the children in France present severe and specific language and/or reading disorders, despite regular remedial therapies. Few studies have measured the effectiveness of treatment administered in a specialist unit. PATIENTS AND METHOD: The aims of this study, focusing on children diagnosed as dysphasic and/or dyslexic, were: During the academic year 2001-2002, 31 children (18 dyslexic and 13 dysphasic) were attending school in our unit. The teaching program and intensive speech therapy (3 hours/week) were tailored for each child according to his/her specific disorders. Reading, spelling and numeracy developmental skills of each child were evaluated by appropriate tools at the beginning and at the end of the year. Impairment was defined by measuring the gap between the observed and the expected skills, according to each child's age. Using a self-control method, progress achieved by each child throughout the year was calculated with each tool, in each subject, by subtracting the impairments disclosed at the beginning from those disclosed at the end of the year. Progression was classified within three groups according to the progress normally expected over an academic year (i.e. nine months) from children with no disabilities attending school regularly; a progression fewer than three months was considered as no progression. Uni- and multivariate analyses including age (< or= or />9), type of pathology (dysphasia/dyslexia), and intellectual quotient (IQ) as covariates was carried out to search for independent prognosticators. RESULTS: The entire group demonstrated during the year significant progress for reading (p = 0.0001), spelling (p = 0.0001) and numeracy (p = 0.0001). Nineteen children (61 percent) showed more progress in reading than normally expected over nine months. Out of the remaining 12 children, 10 demonstrated more progress in spelling and/or numeracy than normally expected over nine months. All three reading evaluation tools disclosed a progression although one was less efficient (p = 0.05). Multivariate analysis disclosed age< or=9 and dysphasia as independent progress prognosticators. CONCLUSION: Placement in a specialist unit allows children suffering from severe dyslexia and dysphasia to lessen the gap in reading, spelling and numeracy. The two prognosticators disclosed highlight the importance of early diagnosis (i.e. before nine years old) and treatment of specific language and/or reading disorders.

Severe cognitive impairment in DMD: obvious clinical indication for Dp71 isoform point mutation screening.

Duchenne muscular dystrophy is associated with variable degrees of selective cognitive defect with lower scores for verbal intelligence and reading abilities. A number of findings have shown that rearrangements located in the second part of the gene seem to be preferentially associated with cognitive impairment. Several dystrophin transcripts are expressed in the brain. The more distal of them, Dp71, is predominant. We have carried out a mutational analysis of Dp71 transcript in 12 DMD patients severely, mildly or not retarded, all without detectable deletion or duplication. We have detected five point mutations causing Dp71 premature translation termination. All were found among the more severely mentally retarded patients of this group (VIQ < 50 and/or no reading acquisition).

Membrane dynamics in human leukemia and lymphoma cells. pH dependency of diphenylhexatriene fluorescence polarization.

Membrane dynamics of human leukemia and lymphoma cell lines were analyzed by investigating the effect of pH on fluorescence polarization (P) of the lipophilic probe diphenylhexatriene (DPH). The degree of P varied as a function of pH, depending on the cell lines. These variations were not detected in phospholipid vesicles. In addition, they were prevented by treatments with glutaraldehyde, sodium azide or phenylmethylsulfanyl fluoride, a specific protease inhibitor. Therefore, these P value changes might be influenced by protein modification.