A systems biology approach for elucidating the interaction of curcumin with Fanconi anemia FANC G protein and the key disease targets of leukemia.

Fanconi anemia (FA) is an autosomal recessive disorder with a high risk of malignancies including acute myeloid leukemia and squamous cell carcinoma. There is a constant search out of new potential therapeutic molecule to combat this disorder. In most cases, patients with FA develop haematological malignancies with acute myeloid leukemia and acute lymphoblastic leukemia. Identifying drugs which can efficiently block the pathways of both these disorders can be an ideal and novel strategy to treat FA. The curcumin, a natural compound obtained from turmeric is an interesting therapeutic molecule as it has been reported in the literature to combat both FA as well as leukemia. However, its complete mechanism is not elucidated. Herein, a systems biology approach for elucidating the therapeutic potential of curcumin against FA and leukemia is investigated by analyzing the computational molecular interactions of curcumin ligand with FANC G of FA and seven other key disease targets of leukemia. The proteins namely DOT1L, farnesyl transferase (FDPS), histone decetylase (EP3000), Polo-like kinase (PLK-2), aurora-like kinase (AUKRB), tyrosine kinase (ABL1), and retinoic acid receptor alpha (RARA) were chosen as disease targets for leukemia and modeled structure of FANC G protein as the disease target for FA. The docking investigations showed that curcumin had a very high binding affinity of -8.1 kcal/mol with FANC G protein. The key disease targets of leukemia namely tyrosine kinase (ABL1), aurora-like kinase (AUKRB), and polo-like kinase (PLK-2) showed that they had the comparable binding affinities of -9.7 k cal/mol, -8.7 k cal/mol, and -8.6 k cal/mol, respectively with curcumin. Further, the percentage similarity scores obtained from PAM50 using EMBOSS MATCHER was shown to provide a clue to understand the structural relationships to an extent and to predict the binding affinity. This investigation shows that curcumin effectively interacts with the disease targets of both FA and leukemia.

Antagonistic molecular interactions of photosynthetic pigments with molecular disease targets: a new approach to treat AD and ALS.

Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are progressive neurodegenerative diseases that affect the neurons in the brain and the spinal cord. Neuroinflamation and apoptosis are key players in the progressive damage of the neurons in AD and ALS. Currently, there is no drug to offer complete cure for both these diseases. Riluzole is the only available drug that can prolong the life time of the ALS patients for nearly 3 months. Molecules that offer good HIT to the molecular targets of ALS will help to treat AD and ALS patients. P53 kinase receptor (4AT3), EphA4 (3CKH) and histone deacetylase (3SFF) are the promising disease targets of AD and ALS. This paper discusses on a new approach to combat neurodegenerative diseases using photosynthetic pigments. The docking studies were performed with the Autodock Vina algorithm to predict the binding of the natural pigments such as ß carotene, chlorophyll a, chlorophyll b, phycoerythrin and phycocyanin on these targets. The ß carotene, phycoerythrin and phycocyanin had higher binding energies indicating the antagonistic activity to the disease targets. These pigments serve as a potential therapeutic molecule to treat neuroinflammation and apoptosis in the AD and ALS patients.

A Novel Technique of Synthesis of Highly Fluorescent Carbon Nanoparticles from Broth Constituent and In-vivo Bioimaging of C. elegans.

Here we have demonstrated a novel single step technique of synthesis of highly fluorescent carbon nanoparticles (CNPs) from broth constituent and in vivo bioimaging of Caenorhabditis elegans (C. elegans) with the synthesized CNPs has been presented. The synthesized CNPs has been characterized by the UV-visible (UV-Vis) absorption spectroscopy, transmission electron microscopy (TEM) and Raman studies. The sp (2) cluster size of the synthesized samples has been determined from the measured Raman spectra by fitting it with the theoretical skew Lorentzian (Breit-Wigner- Fano (BWF)) line shape. The synthesised materials are showing excitation wavelength dependent tunable photoluminescence (PL) emission characteristics with a high quantum yield (QY) of 3 % at a very low concentration of CNPs. A remarkable increase in the intensity of PL emission from 16 % to 39 % in C. elegans has also been observed when the feeding concentration of CNPs to C. elegans is increased from 0.025 % to 0.1 % (w/v). The non-toxicity and water solubility of the synthesized material makes it ideal candidate for bioimaging.

Contemporary and futuristic views of pollution control devices in foundries.

Foundry practices are used in contemporary world to produce large volume of components and products. Foundry practices involve the melting of metals and pouring the molten metal into the cavities called molds. On solidification, the metals which assume the shape of molds are removed as castings. Foundries that employ these practices were growing in large number till the middle part of the twentieth century in the world. After the middle part of the twentieth century, the world community begun to realize that, foundries were emitting pollutants which were affecting the health of humans. In order to overcome this situation, several countries in the world promulgated laws stipulating the maximum level of pollutants that can emit by foundries. These laws affected the functioning and growth of foundries. In order to sustain amidst these constraints, foundries begun to install energy efficient melting technologies and pollution control devices (PCDs). In this back ground, this paper reports to assess the contemporary scenario and project the future needs for sustaining the foundries. During the conduct of this literature review, it was discernable that, research papers have reported three categories of researches. In the first category of research papers, the researches reporting the achievement of cleaner production technologies in foundries using PCDs have appeared. In the second category of research papers, the application of cleaner production technology in foundries located in different countries has been examined. In the third category of research papers, the application of efficient melting technologies and PCDs in different clusters of foundries located in different parts of world has been explored. Subsequently implementation technics of Environmental Management System in cleaner production technics in foundries has been described the analysis of the information and knowledge drawn from these three categories of papers has revealed that, researches exploring the sustenance of foundries situated in different parts of world are required to be carried out intensively in future. The outcome of these researchers will be useful to apply the cleaner production technologies that would be suitable for implementation in different foundry clusters to suit the different conditions prevailing with regard to the adoption of efficient melting technologies and PCDs.

Comparison of Rate of Canine Retraction and Anchorage Potential between Mini-implant and Conventional Molar Anchorage: An In vivo Study.

AIM: The aim of this study was to compare the rate of canine retraction, the anchorage loss, and the change in the inclination of the first molars between molar and mini-implant anchorage. OBJECTIVE: (1) To compare the rate of canine retraction between conventional molar anchorage and mini-implant anchorage in the maxilla and mandible. (2) To compare the amount of anchor loss between mini-implant-anchored and molar-anchored sides during canine retraction in the maxilla and mandible. MATERIALS AND METHODS: Ten patients were included in the study. The implants were loaded immediately by applying a force of 100 g. Measurements were made in the pre-retraction and post-retraction lateral cephalograms. A line drawn vertically from the sella-nasion plane through the distal pterygomaxillary point was used as a reference line. RESULTS: The mean rates of canine retraction were 0.95 and 0.82 mm/month in maxilla on the implant and molar sides, respectively, and were 0.81 and 0.76 mm/month in mandible on the implant and molar sides, respectively. The mean anchorage loss was 0.1 mm on the implant side and 1.3 mm on the molar side of the maxilla and 0.06 mm on the implant side and 1.3 mm on the molar side of the mandible. The mean change in molar inclination was 0.3° on implant side and 2.45° on molar side of the maxilla and was 0.19° on implant side and 2.69° on molar side of the mandible. CONCLUSIONS: Implant anchorage is an efficient alternative to molar anchorage.

Comparison of surface roughness of enamel and shear bond strength, between conventional acid etching and erbium, chromium-doped: Yttrium scandium-gallium-garnet laser etching - An in vitro study.

BACKGROUND: The purpose of the study was to evaluate and to compare the shear bond strength (SBS), adhesive remnant index, and surface roughness of the samples bonded after etching with phosphoric acid and erbium, chromium-doped: Yttrium scandium-gallium-garnet (Er, Cr: YSGG) laser. MATERIALS AND METHODS: In the present analytical/descriptive study, 90 premolars extracted for orthodontic purposes were used, out of which 75 were randomly divided into five groups where five different methods were used to prepare the enamel for bonding; etching with 37% phosphoric acid for 15 s, irradiation with Er, Cr: YSGG laser at 1 watt for 10 s and 20 s, and irradiation with Er, Cr: YSGG laser at 1.5 watt for 10 s and 20 s. Following this, metal brackets were bonded with Transbond XT. Brackets were debonded 24 h, later and SBS were measured, and adhesive remnant index scores were measured. The remaining 15 teeth were used for surface evaluation of these five groups using three-dimensional optical profiler. The results of the SBS testing, adhesive remnant index) scores, and surface roughness values were analyzed by one-way analysis of variance and Tukey honestly significant difference tests with a significant level at 0.05. RESULTS: The difference in bond strength between the laser (1.5 W/20 s) and conventional acid etching was not statistically significant (P > 0.05). For acid etch tech, it was 10.48 Mpa and Laser etch at 1.5 W/20 s 10.46 Mpa bond strength attained by the other groups (1 W/10 Hz, 1 W/20 Hz, and 1.5 W/10 Hz) was significantly less than acid etched, and laser etched (1.5 W/20 Hz) groups with P > 0.05. The surface roughness was found to be similar between the laser- (1.5 W/20 s) and acid-etched groups (P > 0.05). CONCLUSION: Irradiation with 1.5 W/20 s Er, Cr: YSGG laser produced bond strength comparable to acid etching.

Does Change in Thread Shape Influence the Pull Out Strength of Mini Implants? An In vitro Study.

INTRODUCTION: Mini implants form a valuable source for absolute anchorage thereby helping in achieving ideal treatment outcome. Stability of the mini implant is one of the important factors affecting the success of mini implants. Thread shape is a critical factor in the engineering design of mini implant, which affects the primary stability. AIM: To evaluate the effects of thread shape on the pull out strength of mini implants. MATERIALS AND METHODS: Mini implants of five different designs in thread shape (reverse buttress, buttress, 75° joint profile with flutes, trapezoidal and trapezoidal fluted) were used with 10 screws in each group. The mini implants were loaded on to the polyurethane foam block (Sawbones pacific research lab, USA) perpendicular to the surface and the pull out strength was tested using the Instrom testing machine. The control group consisted of mini implants with reverse buttress thread shape. One-way ANOVA and Tukey post-hoc tests were used to compare the pull out strength of the mini implants within as well as between the different groups. RESULTS: The mean in the pull out tests ranged from 13.45 N (trapezoidal) to 61 N (trapezoidal fluted). The tukey post-hoc tests showed a statistically significant difference of 34.5 N between the control group and the trapezoidal fluted group. The level of statistical significance showed p< 0.05. CONCLUSION: Trapezoidal fluted mini implants showed the highest pull out strength when compared to mini implants with other thread designs used in this study. Further studies with the use of Finite Element Method (FEM) and foam blocks of higher density would be required to evaluate the performance of this new thread design.

Assessment of Regeneration of Bone in the Extracted Third Molar Sockets Augmented Using Xenograft (CollaPlugTN Zimmer) in Comparison with the Normal Healing on the Contralateral Side.

INTRODUCTION: Alveolar bone resorption is a significant clinical problem. Bone loss in third molar region following extraction or surgical removal not only leads to periodontal problems in second molar region but also it may lead to some serious problems like increased incidence of angle fractures. In order to reduce the risks following third molar surgery, the socket should be augmented with bone grafts. In recent days guided tissue regeneration is the most accepted and successful technique followed many authors and its efficacy has been proved. MATERIALS AND METHODS: Based upon our clinical experience, the use of bio absorbable collagen wound dressing such as CollaPlugTN has achieved quick healing and more primary wound coverage. Amongst the graft materials collagen is preferable due to its high biocompatibility and hemostatic ability. This study was done to assess the regeneration of bone in the extracted third molar sockets using xenograft (CollaPlugTN-Zimmer) which was compared with the normal healing on the contra lateral side. The assessment was done to analyze post-operative healing complications and to compare the bone density formed between control site and implant site radiologically. CONCLUSION: On this basis of this study, the use of collaplugTN appears to be beneficial to the patient in postoperative wound healing and also for better bone formation. The use of this material was advantageous because of its simplicity of application cost effectiveness and availability. There is enhanced wound healing and early bone formation.

Effects of oral dosing paradigms (gavage versus diet) on pharmacokinetics and pharmacodynamics.

In cancer chemopreventive studies, test agents are typically administered via diet, while the preclinical safety studies normally employ oral gavage dosing. Correspondence in pharmacokinetic and pharmacodynamic profiles between the two dosing approaches cannot be assumed a priori. Sulindac, a non-steroidal anti-inflammatory agent with potential chemopreventive activity, was used to assess effects of the two oral dosing paradigms on its pharmacokinetics and pharmacodynamics. Time-dependent concentrations of sulindac and its sulfone metabolite were determined in plasma and potential target organ, mammary gland. Prostaglandin E(2) was used as a pharmacodynamic biomarker and measured in mammary gland. An inverse linear relationship was detected between pharmacodynamic and pharmacokinetic markers, area under the curve for prostaglandin E(2) levels and sulindac sulfone concentrations, respectively, in the mammary tissue. Marked differences in pharmacokinetics and pharmacodynamics were observed after administration of sulindac by the two oral dosing paradigms. In general, oral gavage resulted in higher peak and lower trough concentrations of sulindac in plasma and mammary tissue, higher area under concentration-time curve in plasma and mammary tissue, and greater effect on prostaglandin E(2) levels than the corresponding diet dosing. This study illustrates potential pitfalls and limitations in trying to generalize based on data obtained with different oral dosing schemes and their extrapolation to potential efficacy and health risks in humans.

Elevated thymic and serum galactosyltransferase with low-molecular-weight acceptor activity in murine leukemia.

Galactosyltransferase activity (UDP-galactose:N-acetyl-D-glucosamine-D-galactosyltransferase) could be measured in thymus and sera from different strains of mice. Total thymic homogenates or thymocyte preparations obtained from thymoma carrying AKR/J mice exhibited higher enzyme activity compared to nonleukemic control mice. A similar difference was also noted in Swiss mouse thymus which develop thymic leukemia upon a single injection of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboximide. Galactosidase, which was 25 times less active than galactosyltransferase, was not responsible for this difference. These observations were extended to an evaluation of the serum level of the enzyme as a potential tumor biomarker. A 3- to 4-fold increase in the activity of galactosyltransferase was detected in serum samples obtained from both leukemic mice models (AKR/J and Swiss) compared to the controls, whereas the sera from P388 tumor-bearing DBA/2 mice showed a statistically nonsignificant increase of only 20%. The data indicate that serum galactosyltransferase (that accepts the low-molecular-weight acceptor, N-acetyl-D-glucosamine) levels are elevated in the presence of thymic leukemia, and suggest the possibility of shedding of this enzyme from the tumor cells to the systemic circulation of the host. The implications, including the potential diagnostic significance of the results, are discussed.

Inhibition of the lectin-induced mitogenic response of thymocytes by glycolipids.

Exogenous gangliosides at concentrations found in serum inhibit the concanavalin A- (Con A) induced mitogenic response of mouse thymocytes. Of four gangliosides tested, the trisialoganglioside, GT1, was the most potent inhibitor. Ceramides, cerebrosides, and sialic acid were not inhibitory at any concentration tested. The inhibition by gangliosides was not due to interference with Con A binding as shown by direct binding studies with [3H]acetyl-Con A nor was it due to a nonspecific killing effect. Thymocytes exposed to a ganglioside concentration 5 times that required to inhibit mitogenesis were still capable of excluding trypan blue up to 44 hr after ganglioside addition. Furthermore, ganglioside inhibition could be reversed by washing the cells 4 hr after addition of the glycolipid. A productive interaction with Con A occurs in the presence of ganglioside as shown by a Con A-induced increase in carbohydrate metabolism. However, uridine and thymidine incorporation are inhibited by the presence of ganglioside. Complete inhibition could be achieved if the glycolipid were added as late as 24 to 28 hr after the Con A in a 48-hr mitogenic assay. The results are discussed in light of recent findings that elevated levels of gangliosides are found in in the sera of tumor-bearing animals, and it is suggested that gangliosides shed by tumor cells could be involved in the generalized immunosuppression observed in such animals.

Implant-based overdenture: A review in patient perspective.

A review in affected person's attitude in abstract care of edentulous patients has to be a priority in elderly individuals. The development of complete dentures (CDs) has been the selection of remedy retaining in mind the socioeconomic popularity, age, and nutritional elements. However, most of the patients complain of loss of retention in mandibular implant-supported overdentures (ODs), which compensated the downside of the loss of retention in complete dentures (CDs). Moreover, implant supported over dentures (ISOVDs) supplied accurate exceptional of lifestyles, esthetics, progressed nutritional deficiencies, and provided good patient satisfaction. The place of dental implants and desire of retentive attachments for implant supported mandibular over dentures (ISOVD) are selected on clinician preference and professional opinion. This text offers a fundamental statistics regarding implant placement, mode of treatment to be selected, and patient care. Two implants provide extraordinary long-term achievement and survival with improved oral capabilities. Single midline implant OD is costly, powerful, and may be a promising alternative. In maxilla, 4-6 implants splinted with bar have located to give true results.

Adjunctive aids for the detection of oral premalignancy.

Early detection of cancer greatly decreases the morbidity and mortality rates and thereby increases the 5-year survival rates. In developing countries like India where the disease is highly prevalent focus is mainly on decreasing the mortality rates which can be easily achieved by detection at an asymptomatic stage. Visual examination has been the standard screening method for screening oral cancer through several decades, and it is well known that conventional visual examination is limited to subjective interpretation and cannot be easily achieved in certain anatomical sites. As a solution to all these adjunctive techniques have emerged, and it has been widely used. An effort is made through this paper to review the most commonly used adjunctive aids for the detection of premalignancy and cancer.

A high-affinity (Ca2+ + Mg2+)-ATPase in plasma membranes of rat ascites hepatoma AH109A cells.

The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity (Ca2+ + Mg2+)-ATPase, EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity (Ca2+ + Mg2+)-ATPase had an apparent half saturation constant of 77 +/- 31 nM for free calcium, a maximum reaction velocity of 9.9 +/- 3.5 nmol ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 microM free calcium. The high-affinity (Ca2+ + Mg2+)-ATPase was absolutely dependent on 3-10 mM magnesium and the pH optimum was within physiological range (pH 7.2-7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent Km of 30 microM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K+, Na+, ouabain, KCN, dicyclohexylcarbodiimide and NaN3, had no significant effect on the activity of high-affinity (Ca2+ + Mg2+)-ATPase. Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 microM. The high-affinity (Ca2+ + Mg2+)-ATPase was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetra-acetic acid. Since the kinetic properties of the high-affinity (Ca2+ + Mg2+)-ATPase showed a close resemblance to those of erythrocyte plasma membrane (Ca2+ + Mg2+)-ATPase, the high-affinity (Ca2+ + Mg2+)-ATPase of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.

Glycolipid sialyltransferases in normal and neoplastic murine thymocytes.

Total labeled glycolipids from thymocytes of leukemic AKR/J mouse thymus incubated for 24 h in the presence of the precursors [3H] galactose or [14C] glucosamine were found to exceed those from non-leukemic thymocytes. The amount of labeled compounds that co-migrated with monosialogangliosides (GM3 and GM2) and disialogangliosides (GD1a and GD1b) was higher, whereas incorporation into monosialoganglioside (GM1) and trisialoganglioside (GT1) was lower in leukemic than in non-leukemic thymocytes. Consistent with this altered pattern of ganglioside distribution was the finding of a higher activity of two of the sialyltransferase activities in homogenates of leukemic thymus as compared to normal thymus. About 2-fold higher activities of the enzymes CMP-AcNeu: lactosylceramide sialyltransferase and CMP-AcNeu: GM1 sialyltransferase were observed in leukemic thymus homogenate compared to homogenates of non-leukemic cells. The substrate affinity of sialyltransferase with GM1 as acceptor from the leukemic thymus was similar to that of the enzyme from normal thymus. Thus, our studies reveal an enzymatic basis for the enhanced rate of synthesis and altered ganglioside profile observed in malignant thymocytes, and are consistent with the general view on the pattern of acidic glycolipids in tumorigenesis.

Identification of the AMP binding sites of rabbit phosphofructo-1-kinase isozymes B and C.

Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo.

Pseudoepitheliomatous Hyperplasia: Relevance in Oral Pathology.

Pseudoepitheliomatous hyperplasia (PEH), a neglected entity by oral pathologist possesses utmost importance in the field of research. Of all the investigative challenges, PEH, a reactive epithelial proliferation is seen secondary to lesions with infectious, inflammatory, reactive, and degenerative origin. Small sized samples, incomplete excision, improper orientation, and dense inflammatory changes render diagnostic confront to the oral pathologist in exclusion of frankly invasive malignant lesions like squamous cell carcinoma from lesions exhibiting PEH. The diagnosis can occasionally be difficult as they mimic other lesions also, on clinic-pathological assessment. Thus, this article gives an insight regarding the various concepts of etiopathogenesis, histopathology, differential diagnosis, and malignant potential of PEH. A combined effort of a clinician and pathologist benefits every patient to rule out malignancy and render appropriate treatment as the only local conservative approach is essential to remove PEH associated lesions.

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors.

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.

Senescence and cytokines modulate the NK cell expression.

We have been investigating senescence-related changes in human peripheral blood natural killer (NK2) cells. Data accumulated so far consistently and clearly show that both basal and cytokine (IL-2 and interferon alpha and gamma) induced antitumor MHC-unrestricted cytotoxic activity of NK cells are well-preserved in the healthy elderly. To investigate if the non-cytotoxic functions of NK cells are also spared from the influence of senescence, recombinant IL-2-inducible secretion of IFN-gamma, which serves as a first line of defense, was examined. The amount of IFN-gamma secreted by purified, 18 h activated NK cells from the elderly was only 25 percent of that released by the cells from the young. Thus, the type 1 cytokine-inducible cytotoxic and cytokine secretory functions appear to be dissociable properties of NK cells, at least in the elderly. However, this aging-related early phase secretory deficit could be overcome by chronic stimulation with IL-2 (7 day culture). Since different subsets could perform different functions, we analyzed the NK subsets by flow cytometry. A minor CD56bright subset and a major CD56dim subset could be distinguished based on the density of expression of the cell surface CD56 molecule (N-CAM). We inquired if immunosenescence is likely to impact the steady-state level of circulating NK subsets. A significant decrease (P < 0.01) in percent CD56bright among CD56+ cells was observed in the elderly with a relative sparing of the CD56dim subset. The CD56bright/CD56dim ratio, perhaps representing NK cell maturity status, declined with age. This maturation-related subset redistribution and partial loss during aging of high affinity IL-2 alpha beta gamma receptor bearing 'immature' CD56bright NK cells has not been reported before. It could be a consequence of the decline in the level of IL-2 during aging. It is concluded that post-adolescent age-associated modulations in human NK cells are not expressed uniformly; they are pronounced in some, subtle in others but negligible in yet other biological parameters.

Immunosenescence of human NK cells: effects on tumor target recognition, lethal hit and interferon sensitivity.

Previous results from this laboratory have shown the preservation of non-MHC-restricted, constitutive oncolytic activity of human peripheral blood NK cells in the elderly as assessed by the chromium release assay which quantitates the lytic endpoint at the cell population level. We have now addressed this senescence-related change at single-cell level using 101 blood samples. Both the efficiency of the initial tumor target binding step i.e., recognition of K562, the NK-sensitive erythroleukemia cell line, as well as the ability of NK cells to deliver lethal hit are highly conserved during healthy aging. In fact, the elderly exhibit a statistically significant, moderately higher frequency of active killers among circulating lymphocytes. Analyzed in another way, a majority of "high NK responders" were found to be older donors, while none in the "low NK responders" group were > 70 years old. Gamma interferon, a gene product as well as an autocrine activator of NK cells, is effective in converting non-lytic "pre-NK" cells to active killers at single-cell level. This in vitro cytokine sensitivity of NK cells is unaltered during immune senescence. The intactness of the NK cell's capacity to be modulated may be vital in both tumor resistance and host viral defenses of aged humans.