Use of 2D FFT and DTW in Protein Sequence Comparison.

Protein sequence comparison remains a challenging work for the researchers owing to the computational complexity due to the presence of 20 amino acids compared with only four nucleotides in Genome sequences. Further, protein sequences of different species are of different lengths; it throws additional changes to the researchers to develop methods, specially alignment-free methods, to compare protein sequences. In this work, an efficient technique to compare protein sequences is developed by a graphical representation. First, the classified grouping of 20 amino acids with a cardinality of 4 based on polar class is considered to narrow down the representational range from 20 to 4. Then a unit vector technique based on a two-quadrant Cartesian system is proposed to provide a new two-dimensional graphical representation of the protein sequence. Now, two approaches are proposed to cope with the varying lengths of protein sequences from various species: one uses Dynamic Time Warping (DTW), while the other one uses a two-dimensional Fast Fourier Transform (2D FFT). Next, the effectiveness of these two techniques is analyzed using two evaluation criteria-quantitative measures based on symmetric distance (SD) and computational speed. An analysis is performed on five data sets of 9 ND4, 9 ND5, 9 ND6, 12 Baculovirus, and 24 TF proteins under the two methods. It is found that the FFT-based method produces the same results as DTW but in less computational time. It is found that the result of the proposed method agrees with the known biological reference. Further, the present method produces better clustering than the existing ones.

MMV method: a new approach to compare protein sequences under binary representation.

In the present work, a new form of descriptor using minimal moment vector (MMV) is introduced to compare protein sequences in the frequency domain under their component wise binary representations. From every sequence, 20 different binary component sequences are formed, each corresponding to 20 amino acids. Each such vector is now shifted from the time domain to the frequency domain by applying the Fast Fourier Transform (FFT). Next, the power spectrum calculated from the FFT values for each component sequence is so normalized that the sum of the components equals 1. The descriptor is defined as a 20-component vector composed of the 20 second-order minimal moments calculated from the normalized spectrum of the 20 component sequences. Once the descriptor is known, the distance matrix is created by applying the Euclidean Distance measure. The phylogenetic tree is generated by applying the unweighted pair group method with the arithmetic mean (UPGMA) algorithm using Molecular Evolutionary Genetics Analysis11 (MEGA11) software. In this work, the datasets used for similarity studies are 9 NADH dehydrogenase 5 (ND5), 12 Baculoviruses, 24 Transferrins (TF) proteins, and 50 Spike Protein of coronavirus. A qualitative measure using rationalized perception is used to compare the effectiveness of the proposed method. Quantitative measure based on symmetric distance (SD) is used to compare the phylogenetic trees of the present method with those obtained by other methods. It is observed that the phylogenetic trees generated by the proposed technique are at par with their known biological references, and they produce results better than those of the earlier methods.Communicated by Ramaswamy H. Sarma.

Choice of Metric Divergence in Genome Sequence Comparison.

The paper introduces a novel probability descriptor for genome sequence comparison, employing a generalized form of Jensen-Shannon divergence. This divergence metric stems from a one-parameter family, comprising fractions up to a maximum value of half. Utilizing this metric as a distance measure, a distance matrix is computed for the new probability descriptor, shaping Phylogenetic trees via the neighbor-joining method. Initial exploration involves setting the parameter at half for various species. Assessing the impact of parameter variation, trees drawn at different parameter values (half, one-fourth, one-eighth). However, measurement scales decrease with parameter value increments, with higher similarity accuracy corresponding to lower scale values. Ultimately, the highest accuracy aligns with the maximum parameter value of half. Comparative analyses against previous methods, evaluating via Symmetric Distance (SD) values and rationalized perception, consistently favor the present approach's results. Notably, outcomes at the maximum parameter value exhibit the most accuracy, validating the method's efficacy against earlier approaches.

PTGAC Model: A machine learning approach for constructing phylogenetic tree to compare protein sequences.

This work proposes a machine learning-based phylogenetic tree generation model based on agglomerative clustering (PTGAC) that compares protein sequences considering all known chemical properties of amino acids. The proposed model can serve as a suitable alternative to the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), which is inherently time-consuming in nature. Initially, principal component analysis (PCA) is used in the proposed scheme to reduce the dimensions of 20 amino acids using seven known chemical characteristics, yielding 20 TP (Total Points) values for each amino acid. The approach of cumulative summing is then used to give a non-degenerate numeric representation of the sequences based on these 20 TP values. A special kind of three-component vector is proposed as a descriptor, which consists of a new type of non-central moment of orders one, two, and three. Subsequently, the proposed model uses Euclidean Distance measures among the descriptors to create a distance matrix. Finally, a phylogenetic tree is constructed using hierarchical agglomerative clustering based on the distance matrix. The results are compared with the UPGMA and other existing methods in terms of the quality and time of constructing the phylogenetic tree. Both qualitative and quantitative analysis are performed as key assessment criteria for analyzing the performance of the proposed model. The qualitative analysis of the phylogenetic tree is performed by considering rationalized perception, while the quantitative analysis is performed based on symmetric distance (SD). On both criteria, the results obtained by the proposed model are more satisfactory than those produced earlier on the same species by other methods. Notably, this method is found to be efficient in terms of both time and space requirements and is capable of dealing with protein sequences of varying lengths.

Protein sequence comparison based on representation on a finite dimensional unit hypercube.

Numerous techniques are used to compare protein sequences based on the values of the physiochemical properties of amino acids. In this work, a single physical/chemical property value based non-binary representation of protein sequences is obtained on a 20 × 20-dimensional unit hypercube. The represented vector expressed in the matrix form is taken as the descriptor. The generalized NTV metric, which is an extension of the NTV metric used for polynucleotide space is taken as a distance measure. Based on this distance measure, a distance matrix is obtained for protein sequence comparison. Using this distance matrix, phylogenetic trees are drawn by using Molecular Evolutionary Genetics Analysis 11 (MEGA11) software applying the neighbor-joining method. Data sets used in this current work are 9-ND4, 9-ND5, 9-ND6, 24 TF-LF proteins, 27 different viruses and 127 proteins from the protein kinase C (PKC) family. Two sets of phylogenetic trees are obtained - one based on property value of polarity and the other based on property value of molecular weight. They are found to be exactly the same. Similar results also hold for other single property value based representation. The present trees are individually tested for efficiency based on the criterion of rationalized perception and computational time. The results of the present method are compared with those obtained earlier by other methods on the same protein sequences using assessment criteria of Symmetric distance (SD), Correlation coefficient, and Rationalized perception. In all the cases, the present results are found to be better than the results of other methods under comparison.Communicated by Ramaswamy H. Sarma.

Mathematical Approach to Protein Sequence Comparison Based on Physiochemical Properties.

The difficult aspect of developing new protein sequence comparison techniques is coming up with a method that can quickly and effectively handle huge data sets of various lengths in a timely manner. In this work, we first obtain two numerical representations of protein sequences separately based on one physical property and one chemical property of amino acids. The lengths of all the sequences under comparison are made equal by appending the required number of zeroes. Then, fast Fourier transform is applied to this numerical time series to obtain the corresponding spectrum. Next, the spectrum values are reduced by the standard inter coefficient difference method. Finally, the corresponding normalized values of the reduced spectrum are selected as the descriptors for protein sequence comparison. Using these descriptors, the distance matrices are obtained using Euclidian distance. They are subsequently used to draw the phylogenetic trees using the UPGMA algorithm. Phylogenetic trees are first constructed for 9 ND4, 9 ND5, and 9 ND6 proteins using the polarity value as the chemical property and the molecular weight as the physical property. They are compared, and it is seen that polarity is a better choice than molecular weight in protein sequence comparison. Next, using the polarity property, phylogenetic trees are obtained for 12 baculovirus and 24 transferrin proteins. The results are compared with those obtained earlier on the identical sequences by other methods. Three assessment criteria are considered for comparison of the results-quality based on rationalized perception, quantitative measures based on symmetric distance, and computational speed. In all the cases, the results are found to be more satisfactory.

Nonobese population in a developing country has a high prevalence of nonalcoholic fatty liver and significant liver disease.

UNLABELLED: There is a paucity of community-based epidemiological data on nonalcoholic fatty liver (NAFL) among nonaffluent populations in developing countries. Available studies are radiological and/or biochemical and lack histological assessment, limiting their strength. We conducted a prospective epidemiological study comprising a 1:3 subsample of all adult (>18 years) inhabitants of a rural administrative unit of West Bengal, India. Subjects positive for hepatitis B virus and/or hepatitis C virus infection and consuming any amount of alcohol were excluded. Diagnosis of NAFL was by dual radiological screening protocol consisting of ultrasonographic and computed tomographic examination of the liver. Transient elastographic examination and liver biopsy were performed in a subset to identify significant liver disease. The risk factors of having NAFL were analyzed. A total of 1,911 individuals were analyzed, 7% of whom were overweight and 11% of whom had abdominal obesity. The prevalence of NAFL, NAFL with elevated alanine aminotransferase, and cryptogenic cirrhosis was 8.7%, 2.3%, and 0.2%, respectively. Seventy-five percent of NAFL subjects had a body mass index (BMI) <25 kg/m(2), and 54% were neither overweight nor had abdominal obesity. The subjects with the highest risk of having NAFL were those with a BMI >25 kg/m(2) (odds ratio 4.3, 95% confidence interval 1.6-11.5). Abdominal obesity, dysglycemia (fasting plasma glucose >100 mg/dL or elevated homeostatic model assessment of insulin resistance), and higher income were the other risk factors. Even having a normal BMI (18.5-24.9 kg/m(2)) was associated with a 2-fold increased risk of NAFL versus those with a BMI <18.5 kg/m(2). CONCLUSION: There is a significant prevalence of NAFL and potentially significant liver disease, including cryptogenic cirrhosis, in this predominantly nonobese, nonaffluent population in a developing country. NAFL will be a major determinant of future liver disease burden in countries of the developing world.

Hepatic steatosis is associated with cardiometabolic risk in a rural Indian population: A prospective cohort study.

BACKGROUND/OBJECTIVES: While adiposity and hepatic steatosis are linked to cardiovascular risk in developed countries, their prevalence and impact in low-income countries are poorly understood. We investigated the association of anthropomorphic variables and hepatic steatosis with cardiometabolic risk profiles and subclinical cardiovascular disease (CVD) in a large rural Indian cohort. METHODS: In 4691 individuals in the Birbhum Population Project in West Bengal, India, we performed liver ultrasonography, carotid ultrasound and biochemical and clinical profiling. We assessed the association of hepatic steatosis and anthropomorphic indices (BMI, waist circumference) with CVD risk factors (dysglycemia, dyslipidemia, hypertension) and subclinical CVD (by carotid intimal-medial thickness). RESULTS: Rural Indians exhibited a higher visceral adiposity index and pro-atherogenic dyslipidemia at a lower BMI than Americans. Individuals with any degree of hepatic steatosis by ultrasound had a greater probability of dysglycemia (adjusted odds ratio, OR=1.67, 95% CI 1.31-2.12, P<0.0001) and pro-atherogenic dyslipidemia (OR=1.33, 95% CI 1.07-1.63, P=0.009). We observed a positive association between liver fat, adiposity and carotid intimal-medial thickness (CIMT) in an unadjusted model (ß=0.02, P=0.0001); the former was extinguished after adjustment for cardiometabolic risk factors. CONCLUSIONS: In a large population of rural Indians, hepatic steatosis and waist circumference were associated with prevalent cardiometabolic risk and subclinical CVD at lower BMI relative to multi-ethnic Americans, though the association of the former with subclinical CVD was extinguished after adjustment. These results underscore the emerging relevance of hepatic steatosis and adiposity in the developing world, and suggest efforts to target these accessible phenotypes for cardiometabolic risk prevention.

Detection of immune-complexed 9-O-acetylated sialoglycoconjugates in the sera of patients with pediatric acute lymphoblastic leukemia.

Although childhood acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, reliable techniques are needed to determine treatment outcome. Over expression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) on lymphoblasts and concomitant anti-9-OAcSGs was found to have a diagnostic and prognostic potential. However, the presence of circulatory immune-complexed antigens remains unknown. The present study was aimed to evaluate whether immune-complexed 9-OAcSGs can be harnessed for better disease management. Immune-complexed antigens were evaluated in ALL sera (n=262) by a Dot-blot using a 9-OAcSAalpha2-6GalNAc-specific lectin, Achatinin-H. Using three serum samples, the inter- and intra-assay imprecision was evaluated as 11-13% and 7-11%, respectively. The recovery of spiked 9-OAcSGs was 84.2-95.4%. The central 95% reference interval for immune-complexed 9-OAcSGs in normal human sera (NHS, n=144) was 2.9-3.4 mug/ml irrespective of sex and age. At disease presentation, the immune-complexed 9-OAcSGs were fivefold higher than NHS, decreased with remission induction and importantly, reappeared with clinical relapse. Sera from patients with other hematological disorders (n=86) showed negligible levels. The Dot-blot demonstrated the potential application of immune-complexed antigen as a disease-specific marker and its efficacy as a sensitive and specific method that could serve as an economical yet effective index for monitoring disease status.

Antibodies against 9-O-acetylated sialoglycans: a potent marker to monitor clinical status in childhood acute lymphoblastic leukemia.

BACKGROUND: Although childhood acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, reliable techniques are needed to determine treatment outcome and predict impending relapse. In ALL, the cell surface over expression of 9-O-acetylated sialoglycans (9-OAcSGs) on lymphoblasts and concomitant high antibody titers in patients' sera was reported. OBJECTIVES: The present study was aimed to evaluate whether anti-9-OAcSG titers can be harnessed to monitor the clinical outcome of ALL. DESIGN AND METHODS: Anti-9-OAcSGs were analyzed by ELISA in children receiving either UK ALL X (n = 69, Group I) in India or UK ALL 97 (n = 47, Group II) in UK along with age-matched normal healthy controls at different time points over a period of >2 years. An attempt was also made to investigate subclass distribution of disease-specific IgG. Moreover, 17 patients having a higher sample size were longitudinally monitored. RESULTS: Antibody levels were raised at disease presentation, decreased with remission induction, and importantly, reappeared with clinical relapse. Sera from patients with other hematological disorders and normal controls showed negligible levels of circulating anti-9-OAcSGs. In patients of both Groups I and II, the assay showed high sensitivity (98.92% and 96.77%) and specificity (92.1% and 95.91%), respectively. IgG subclass analyses during different phases of treatment revealed that 9-OAcSG-specific IgG(1) could serve as a better prognostic marker in ALL. CONCLUSIONS: This study demonstrated the potential of this disease-specific antibody as an alternate marker in diagnosis and long-term assessment of ALL patients, suggesting its application in detection and prediction of impending relapse. Therefore, the expression of anti-9-OAcSGs, irrespective of their treatment protocol, may serve as an economical yet effective index for monitoring of childhood ALL.

Interaction of different hemoglobinopathies in Eastern India with a view to establish genotype-phenotype correlation.

Analysis of the molecular basis of hemoglobinopathies provides an opportunity to define genotype-phenotype variations as well as establish the origin of mutation. The present study deals with a large cohort of 1,661 cases referred to the counseling unit and 889 individuals from random screening of the population of Tripura. Characterization of mutation in 291 cases (582 alleles) was performed by the PCR-ARMS method using genomic DNA. The haplotype of 56 beta(E) mutation-bearing chromosomes were identified by the RFLP-PCR method. Genotypes were constructed and correlated with hematological and clinical phenotypes. IVS-1nt 5 (G-->C) mutation was observed as the most frequent mutation, followed by codon 30 (G-->C). Production of HbE was significantly (P < 0.001) higher in nontransfusion-dependent Ebeta-thalassemia patients. beta(E) mutation was observed only on four haplotypes linked to framework 2. Type 2 haplotype was observed mainly from chromosomes of Tripura origin, but none from South Bengal. Homozygous E individuals with 1//1 genotype were significantly (P < 0.01) more anemic compared to individuals with 2//2 genotype. This work creates a database of hemoglobinopathy mutations for the population of Eastern India which will facilitate prenatal diagnosis and counseling. Am. J. Hum. Biol. 12:454-459, 2000. Copyright 2000 Wiley-Liss, Inc.

O-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL).

Exploiting the selective affinity of Achatinin-H towards 9-O-acetylneuraminic acid(alpha2-6)GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on hematopoietic cells of children suffering from acute lymphoblastic leukemia (ALL), indicative of defective sialylation associated with this disease. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using several synthetic sialic acid analogues. They are functionally active signaling molecules as demonstrated by their role in mediating lymphoproliferative responses and consequential increased production of IFN-gamma due to specific stimulation of Neu5,9Ac(2)-GPs on PBMC(ALL) with Achatinin-H. Cells devoid of 9-O-acetylations (9-O-AcSA(-)) revealed decreased nitric oxide production as compared to 9-O-AcSA(+) cells on exposure to IFN-gamma. Under this condition, a decrease in viability of 9-O-AcSA(-) cells as compared to 9-O-AcSA(+) cells was also observed which was reflected from increased caspase 3 activity and apoptosis suggesting the protective role of this glycotope. These Neu5,9Ac(2)-GPs are also capable of inducing disease-specific anti-Neu5,9Ac(2)-GPs antibodies in ALL children. Additionally, we have observed that disease-specific anti-Neu5,9Ac(2)-GPs have altered glycosylation profile, and they are incapable of exerting a few Fc-glycosylation-sensitive effector functions. These observations hint toward a disbalanced homeostasis, thereby enabling the cancer cells to escape host defense. Taken together, it may be hypothesized that Neu5,9Ac(2)-GPs and their antibodies play a prominent role in promoting the survival of lymphoblasts in ALL.

Altered erythrocyte membrane characteristics during anemia in childhood acute lymphoblastic leukemia.

Anemia is a prominent feature in children with acute lymphoblastic leukemia (ALL). To investigate the erythrocyte features during anemia in these patients, we studied the altered characters of these cells and oxidative stress imposed in their serum. This investigation reveals that erythrocytes from ALL patients show (1) increased membrane fluidity detected by fluorescence anisotropy studies, increased osmotic fragility detected by hemolysis of erythrocytes in different saline concentrations, and increased hydrophobicity as measured by binding with 8-anilino-1-naphthalenesulfonic acid, (2) enhanced (approximately threefold) glycosylation and sialylation, monitored by digoxigenin enzyme assay, and (3) expression of disease-specific 210, 105, 83, 54, and 28 kDa 9-O-acetyl sialoglycoconjugates (9-O-AcSGs) demonstrated by Western blot analysis and fluorescence-activated cell sorter (FACS) analysis studies using Achatinin-H with specificity towards 9-O-AcSAalpha2-6GalNAc as the analytical probe. (4) In addition, induced oxidative stress was observed in the sera of these children as indicated by increased nitric oxide (approximately fourfold) and thiobarbituric acid (TBA) reactive species (twofold) as detected by Griess reaction and TBA assay, respectively. For all the experiments, erythrocytes from normal individuals served as controls. Thus, the altered membrane characteristics together with their exposure to induced oxidative stress in serum are found to be a few features restricted to diseased erythrocytes. Taken together, our results are suggestive of their interplay in the contribution to the observed anemia in these patients, which may be exploited for better management of the disease.

Interferon gamma promotes survival of lymphoblasts overexpressing 9-O-acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (ALL).

An enhanced linkage-specific 9-O-acetylated sialic acid (9-O-AcSA) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia, ALL (PBMC(ALL), 9-O-AcSA+ cells) was demonstrated by using a lectin, Achatinin-H, whose lectinogenic epitope was 9-O-AcSAalpha2-6GalNAc. Our aim was to evaluate the in vitro contributory role of this glycotope (9-O-AcSAalpha2-6GalNAc) towards the survival of these 9-O-AcSA+ cells in ALL patients. For direct comparison, 9-O-AcSA- cells were generated by removing O-acetyl group of 9-O-AcSA present on PBMC(ALL) using O-acetyl esterase. An elevated level of serum interferon gamma (IFN-gamma) in affected children led us to think that PBMC(ALL) are continuously exposed specifically to this cytokine. Accordingly, 9-O-AcSA+ and 9-O-AcSA- cells were exposed in vitro to IFN-gamma. A twofold increased NO release along with inducible NO synthase (iNOS) mRNA expression by the 9-O-AcSA+ cells was observed as compared to the 9-O-AcSA- cells. The decreased viability of IFN-gamma exposed 9-O-AcSA- cells as compared to 9-O-AcSA+ cells were reflected from a 5.0-fold increased caspase-3-like activity and a 10.0-fold increased apoptosis in the 9-O-AcSA- cells when production of NO was lowered by adding competitive inhibitor of iNOS in reaction mixture. Therefore, it may be envisaged that a link exists between induction of this glycotope and their role in regulating viability of PBMC(ALL). Taken together, it is reasonable to hypothesise that O-acetylation of sialic acids on PBMC(ALL) may be an additional mechanism that promotes the survival of lymphoblasts by avoiding apoptosis via IFN-gamma-induced NO production.

Increased interferon gamma production by peripheral blood mononuclear cells in response to stimulation of overexpressed disease-specific 9-O-acetylated sialoglycoconjugates in children suffering from acute lymphoblastic leukaemia.

Disease-specific over-expression of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia (ALL, PBMC(ALL)) has been demonstrated using a lectin, Achatinin-H, with specificity towards 9-O-AcSAalpha2-6GalNAc. This study investigated the contributory role of 9-O-AcSGs induced on PBMC(ALL). Stimulation of PBMC(ALL) with Achatinin-H through 9-O-AcSGs led to a lymphoproliferative response with a significantly increased interferon-gamma (IFN-gamma) production when compared with unstimulated cells as demonstrated by enzyme-linked immunosorbent assay and mRNA expression. Under identical conditions, PBMC(ALL) ablated of O-acetylations did not respond to such stimulation. In summary, it may be concluded that stimulation of over-expressed 9-O-AcSGs regulate signalling for proliferation, leading to the release of IFN-gamma. Controlled expression of these molecules may be exploited as potential targets for therapy, promising beneficial effects to children with ALL.

Over-expressed IgG2 antibodies against O-acetylated sialoglycoconjugates incapable of proper effector functioning in childhood acute lymphoblastic leukemia.

Earlier studies have demonstrated an over-expression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) on lymphoblasts, concomitant with high titers of anti-9-OAcSGs in childhood acute lymphoblastic leukemia (ALL). The present study was aimed to evaluate whether this high induction of anti-9-OAcSGs at disease presentation contributes toward immune surveillance. Accordingly, anti-9-OAcSGs were affinity purified from sera of ALL patients and normal individuals, and their specificity toward the glycotope having terminal 9-O-acetylated sialic acid-linked subterminal N-acetyl galactosamine (GalNAc) in alpha2-6 manner (9-OAcSAalpha2-6GalNAc) was established by hemagglutination assay, flow cytometry and confocal microscopy. Subclass distribution of anti-9-OAcSGs revealed a predominance of IgG2 in ALL. Analysis of glycosylation of anti-9-OAcSGs purified from sera of ALL patients (IgG(ALL)) and normal individuals (IgG(N)) by digoxigenin glycan enzyme assay, fluorimetric estimation, gas-liquid chromatography and lectin-binding assays demonstrated that disease-specific antibodies differ in content and nature as compared with normal controls. Enhanced amount of 9-OAcSA-specific IgG2 induced in ALL was unable to trigger activation of FcgammaR, the complement cascade and cell-mediated cytotoxicity, although its glycotope-binding ability remains unaffected. Interestingly, only IgG1N emerged as the potent mediator of cell-mediated cytotoxicity, complement fixation and activator of effector cells through FcgammaR. In ALL, the observed subclass switching of anti-9-OAcSGs to IgG2, alteration in their glycosylation profile along with impairment of a few Fc-glycosylation-sensitive effector functions hints toward a disbalanced homeostasis, thereby evading the host defense. These findings justify further evaluation of the mechanism for functional unresponsiveness of antibodies and production of 9-OAcSA-specific chimeric antibodies with normal Fc domain for therapeutic applications.

Differential expression of 9-O-acetylated sialoglycoconjugates on leukemic blasts: a potential tool for long-term monitoring of children with acute lymphoblastic leukemia.

Earlier studies have demonstrated overexpression of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) on lymphoblasts, concomitant with high titers of anti-9-O-AcSG antibodies in childhood acute lymphoblastic leukemia (ALL). Our aim was to evaluate the correlation between expression of different 9-O-AcSGs during chemotherapeutic treatment. Accordingly, expression of 9-O-AcSGs on lymphoblasts of ALL patients (n = 70) were longitudinally monitored for 6 years (1997-2002), using Achatinin-H, a 9-O-acetylated sialic acid (9-O-AcSA) binding lectin with preferential affinity for 9-O-AcSGs with terminal 9-O-AcSA alpha 2-->6GalNAc. Western blot analysis of patients (n = 30) showed that 3 ALL-specific 9-O-AcSGs (90, 120 and 135 kDa) were induced at presentation; all these bands disappeared after treatment in patients (n = 22) who had disease-free survival. The 90 kDa band persisted in 8 patients who subsequently relapsed with reexpression of the 120 kDa band. FACS analysis revealed that at presentation (n = 70) 90.1 +/- 5.0% cells expressed 9-O-AcSGs, which decreased progressively with chemotherapy, remained <5% during clinical remission and reappeared in relapse (80 +/- 10%, n = 18). Early clearance of 9-O-AcSG(+) cells, during 4-8 weeks of treatment showed a good correlation with low risk of relapse. Sensitivity of detection of 9-O-AcSG(+) cells was 0.1%. Numbers of both high- and low-affinity binding sites were maximum at presentation, decreased with treatment and increased again in clinical relapse. We propose that close monitoring of 90 and 120 kDa 9-O-AcSGs may serve as a reliable index for long-term management of childhood ALL and merits therapeutic consideration.

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia.

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.

Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase-dependent apoptosis in chronic myelogenous leukemic cells.

We report that chlorogenic acid (Chl) induces apoptosis of several Bcr-Abl-positive chronic myelogenous leukemia (CML) cell lines and primary cells from CML patients in vitro and destroys Bcr-Abl-positive K562 cells in vivo. In contrast, this compound has no effect on the growth and viability of Bcr-Abl-negative lymphocytic and myeloid cell lines and primary CML cells. Sodium chlorogenate (NaChl) exhibits 2-fold higher efficiency in killing K562 cells compared with Chl. NaChl also induces growth inhibition of squamous cell carcinoma (HSC-2) and salivary gland tumor cells (HSG), although at 50-fold higher concentration. NaChl inhibits autophosphorylation of p210(Bcr-Abl) fusion protein rapidly. We demonstrate that p38 phosphorylation is increased in Bcr-Abl-positive cells after treatment with NaChl and closely paralleled the inhibition of Bcr-Abl phosphorylation. NaChl did not increase phosphorylation of p38 in Bcr-Abl-negative cells including HSC-2 and HSG that are responsive to this compound, indicating that p38 activation by NaChl is dependent on Bcr-Abl kinase inhibition. Inhibition of p38 activity by SB203580 significantly reduced NaChl-induced apoptosis of K562 cells, whereas activation of p38 by anisomycin augmented the apoptosis. These findings indicate that inhibition of Bcr-Abl kinase leading to activation of p38 mitogen-activated protein (MAP) kinase may play an important role in the anti-CML activity of Chl.