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Steel factor (SF) is a growth and survival factor for hematopoietic cells. The receptor for SF, c-Kit, contains intrinsic tyrosine kinase activity, and binding of SF induces rapid tyrosine phosphorylation of several cellular proteins, including c-Kit itself. Activation of c-Kit is shown here to induce tyrosine phosphorylation of CRKL, and CRKL coprecipitated with c-Kit through an interaction that required the CRKL SH3 domains and not the SH2 domain. CRKL associated with c-Kit indirectly as part of a larger complex of proteins. Two proteins in this complex were identified as the p85 regulatory subunit of phosphatidylinositol 3-kinase (p85(PI3K)) and the proto-oncoprotein p120(CBL). Because p85(PI3K) is known to bind to the activated c-Kit receptor, the possibility that CRKL interacted with c-Kit indirectly through p85(PI3K) was investigated. Far Western blotting with a CRKL-SH3 glutathione S-transferase fusion protein showed that CRKL binds directly to p85(PI3K )in vitro. However, although a small amount of CRKL was preassociated with p85(PI3K), the interaction was increased after SF stimulation, suggesting that the interactions of these three proteins are complex. We conclude that SF induces the formation of a signaling complex potentially containing CRKL and p120(CBL), both of which bind to c-Kit through p85(PI3K). These data suggest that one function of CRKL in normal cells might be to recruit signaling molecules such as CBL into a complex with PI3K. Such complexes could be important in propagating signals involving PI3K such as gene expression and adhesion.

Thrombopoietin (TPO) promotes megakaryocyte growth and development. Its receptor, c-MPL, is restricted to cells of megakaryocytic lineage and stem cells. We have previously shown that activation of c-MPL by thrombopoietin rapidly activates at least two cytoplasmic tyrosine kinases, JAK2 and TYK2, after ligand binding. Phosphatidylinositol-3' kinase (PI3K) has been shown to play an important role in downstream signaling for many receptors. Thrombopoietin was found to also rapidly activate phosphatidylinositol-3' kinase, and the phosphatidylinositol-3' kinase inhibitor wortmannin decreased proliferation of thrombopoietin-stimulated cells, implying that phosphatidylinositol-3' kinase may have a regulatory role in thrombopoietin signaling. In immunoprecipitation studies, the regulatory subunit of phosphatidylinositol-3' kinase, p85PI3K, associated with several tyrosine phosphoproteins, and the major phosphoprotein was a 120 kDa protein identified as p120CBL. The phosphatidylinositol-3' kinase-enzyme activity in p120CBL immunoprecipitates was elevated in thrombopoietin-stimulated cells as compared to immunoprecipitates from unstimulated cells. p120CBL may be involved in signaling pathways activated by c-MPL which involve phosphatidylinositol-3' kinase.

Chronic myelogenous leukemia (CML) and some acute lymphoblastic leukemias (ALL) are caused by the t(9;22) chromosome translocation, which produces the constitutively activated BCR/ABL tyrosine kinase. When introduced into factor dependent hematopoietic cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins. One prominent BCR/ABL substrate is p120CBL, the cellular homolog of the v-Cbl oncoprotein. In an effort to understand the possible contribution of p120CBL to transformation by BCR/ABL, we looked for cellular proteins which associate with p120CBL in hematopoietic cell lines transformed by BCR/ABL. In addition to p210BCR/ABL and c-ABL, p120CBL coprecipitated with an 85 kDa phosphoprotein, which was identified as the p85 subunit of PI3K. Anti-p120CBL immunoprecipitates from BCR/ABL-transformed, but not from untransformed, cell lines contained PI3K lipid kinase activity. Interestingly, the adaptor proteins CRKL and c-CRK were also found in these complexes. In vitro binding studies indicated that the SH2 domains of CRKL and c-CRK bound directly to p120CBL, while the SH3 domains of c-CRK and CRKL bound to BCR/ABL and c-ABL. The N-terminal and the C-terminal SH2 and the SH3 domain of p85PI3K bound directly in vitro to p120CBL. The ABL-SH2, but not ABL-SH3, could also bind to p120CBL. These data suggest that BCR/ABL may induce the formation of multimeric complexes of signaling proteins which include p120CBL, PI3K, c-CRK or CRKL, c-ABL and BCR/ABL itself.

Interleukin-12 (IL-12) is a heterodimeric cytokine that augments the cytolytic activity of human NK cells and T cells but has little mitogenic activity on resting lymphocytes. The intracellular signaling pathways involved in NK cell activation by IL-12 have not been fully characterized. In the present studies we found that IL-12 induces tyrosine phosphorylation of a 56-kDa protein, identified in Western blot experiments as p56lck, in resting NK cells. IL-12 was active in the range of 0.1 to 1000 U/ml, with maximal activity between 10 and 100 U/ml (30 and 300 pM). The maximal effect was noted 5 min after stimulation and was almost completely inhibited by genistein. IL-12 induced similar effects in resting and IL-2 activated NK cells. Following IL-12 stimulation of resting NK cells, immunoprecipitated lck kinase exhibited increased in vitro autophosphorylation activity 5 and 10 min after activation. Several additional substrates were phosphorylated in vitro following IL-12 stimulation, including proteins of 70 and 110 kDa. These studies indicate that lck tyrosine kinase is involved in the IL-12 signaling pathway in human NK cells. As the primary functional effect of IL-12 on resting NK cells is the enhancement of cytolytic activity and secretory function rather than cell proliferation, these findings suggest that lck tyrosine kinase is involved in these signaling pathways in human NK cells.

Macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. In the present studies using human monocytes, we show that M-CSF induces interaction of the Grb2 adaptor protein with the focal adhesion kinase pp125FAK. The results demonstrate that tyrosine-phosphorylated pp125FAK directly interacts with the SH2 domain of Grb2. The findings indicate that a pYENV site at Tyr-925 in pp125FAK is responsible for this interaction. We also demonstrate that the Grb2-FAK complex associates with the GTPase dynamin. Dynamin interacts with the SH3 domains of Grb2 and exhibits M-CSF-dependent tyrosine phosphorylation in association with pp125FAK. These findings suggest that M-CSF-induced signaling involves independent Grb2-mediated pathways, one leading to Ras activation and another involving pp125FAK and a GTPase implicated in receptor internalization.

Proline accumulated in the shoots of seedlings of rice (Oryza sativa), mustard (Brassica juncea) and mung bean (Vigna radiata) exposed to UV radiations. The level of proline in the seedlings increased significantly with increase in UV exposure time. The production of malondialdehyde (an indice of lipid peroxidation) was also higher in the shoots of seedlings exposed to UV radiation as compared to controls, suggesting that UV radiations promote lipid peroxidation. The extent of UV radiation promoted enhancement in the levels of proline as well as that of malondialdehyde was higher in the seedlings of rice than those of mung bean or mustard. This lead us to believe that UV radiation induced proline accumulation protects plants against UV radiation promoted peroxidative processes. UV radiations also promoted peroxidation in linolenic acid micelles. The presence of proline along with linolenic acid micelles during UV exposure caused a considerable reduction in the production of malondialdehyde. This study, for the first time shows that plants exposed to UV radiations accumulate proline and proline can protect plant cells against UV radiation induced peroxidative processes.

The T cell molecule CD28 provides a co-stimulatory signal that is required for T cell proliferation, and has been implicated in the control of T cell anergy. An important clue to the signaling mechanism of CD28 is the finding that CD28 can bind to phosphatidylinositol 3-kinase (PI 3-kinase) by means of a cytoplasmic phospho-YMNM (pYMNM) motif. A remaining issue concerns whether CD28 can recruit other intracellular signaling molecules. In this study, we show that CD28 uses the same pYMNM motif to recruit a second intracellular protein, GRB-2. CD28-associated GRB-2, as detected by anti-GRB-2 immunoblotting, was found in human peripheral T cells, HPB-ALL and Jurkat cells. As in the case of PI3-kinase, antibody-induced cross-linking of CD28 induces a time-dependent recruitment of GRB-2. Likewise, mutation of the pY-191 residue within the pYMNM motif reduces GRB-2 binding. Peptide binding studies show that the SH2 domain of GRB-2 binds to the pYMNM motif with an affinity comparable to GRB-2/SHC, but some 10- to 100-fold lower than the CD28/PI 3-kinase. Despite this, CD28/GRB-2 and CD28/PI 3-kinase complexes are found to co-exist in peripheral T cells. Finally, immunoblotting shows that CD28 also associates with the gene product of the human homolog of the Drosophila Son of sevenless gene (SOS), a GRB-2-complexed guanine nucleotide exchange factor responsible for converting p21ras to a GTP-bound active state. CD28-associated GRB2/SOS is likely to serve an important link in the regulation of p21ras and lymphokine expression mediated by CD28.

CTLA-4 is a T cell antigen that is structurally related to CD28 and serves as a high affinity ligand for the B cell antigen B7-1/2. Unlike CD28, the function of CTLA-4 is unclear, although reports have implicated the antigen in the costimulation of T cells. Recently, phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the costimulatory function of CD28 by virtue of its ability to bind to a pYMNM motif within the cytoplasmic tail of the antigen. In this study, we show that CTLA-4 can also associate with PI 3-kinase as detected by lipid kinase analysis and immunoblotting with anti-p85 antiserum. High pressure liquid chromatographic separation of deacylated lipids showed the presence of a peak corresponding to PI-3-P. Anti-CTLA-4 ligation of the receptor induced a significant increase in the levels of precipitable PI 3-kinase activity. Peptide binding studies revealed that the NH2- and COOH-terminal SH2 domains of p85 bind the CTLA-4 cytoplasmic pYVKM motif with an affinity (ID50: 0.6 and 0.04 microM), that is similar to CD28. CTLA-4 binding to PI 3-kinase provides further evidence that CTLA-4 is not an inert counterreceptor, but rather is coupled to an intracellular signaling molecule with the capacity to regulate cell growth.

Aims: To examine the correlation between bromodeoxyuridine (BrdU) labelling indices (LI) and tubular damage in renal biopsy specimens; to evaluate the diagnostic and prognostic potential of measuring cell proliferation in a variety of renal lesions., Methods: In vitro BrdU labelling of renal biopsy specimens was undertaken and labelled cells were detected in routinely fixed, paraffin wax embedded sections by immunohistochemistry. The BrdU LI were calculated as percentages for the three types of tubular cells--proximal and distal convoluted tubules and medulla (LI/PCT, LI/DCT, LI/Med)--and a total tubular BrdU LI (LI/Tub) was also calculated for each biopsy specimen. Histological features indicative of tubular damage were also scored and a total tubular damage score obtained for each biopsy specimen., Results: The one hour labelling process did not affect tissue morphology or impede subsequent diagnosis. Four biopsy specimens were obtained from three renal transplant recipients. Diagnosis of 19 non-transplant biopsy specimens revealed a variety of renal lesions. Total tubular damage scores ranged from 0 to 25 and the LI/Tub ranged from 0 to 3.68% in all 23 biopsy specimens. Analyses of variance showed highly significant correlations between the total tubular damage score and both LI/Tub (p = 0.004) and LI/PCT (p = 0.004); a weaker correlation was found between the total tubular damage score and LI/DCT (p = 0.013)., Conclusions: A correlation was found between tubular damage and BrdU LI. This was most clearly seen in the proximal tubules. However, as the study was limited to a few examples of specific forms of glomerular or interstitial disease, firm conclusions about the value of BrdU labelling in routine diagnosis and prognosis could not be drawn.

The effect of pranayama a controlled breathing practice, on exercise tests was studied in athletes in two phases; sub-maximal and maximal exercise tests. At the end of phase I (one year) both the groups (control and experimental) achieved significantly higher work rate and reduction in oxygen consumption per unit work. There was a significant reduction in blood lactate and an increase in P/L ratio in the experimental group, at rest. At the end of phase II (two years), the oxygen consumption per unit work was found to be significantly reduced and the work rate significantly increased in the experimental group. Blood lactate decreased significantly at rest in the experimental group only. Pyruvate and pyruvate-lactate ratio increased significantly in both the groups after exercise and at rest in the experimental group. The results in both phases showed that the subjects who practised pranayama could achieve higher work rates with reduced oxygen consumption per unit work and without increase in blood lactate levels. The blood lactate levels were significantly low at rest.

A method is described for the direct detection of lectins, agglutinating erythrocytes, on nitrocellulose membranes after Western blotting, thus avoiding protein extraction from specific bands in the gel, followed by agglutination assays. The methodology essentially involves exposing the lectin band on a nitrocellulose strip to trypsinized rabbit erythrocytes (2%, in 0.15 M NaCl) for 30 min at 37 degrees C and then carefully transferring the membrane to saline (4 degrees C) for a few gentle washes and then fixing it in a solution (0.2% glutaraldehyde in 0.15 M NaCl) for 30 min. Later, the membrane is gently washed several times in 0.15 M NaCl containing 10 mM beta-alanine. The lectin band is visualized as a red agglutinated patch. The method is specific for lectins that can agglutinate red blood cells and virtually has no cross reactivity with the various nonlectin proteins tested. Binding of erythrocytes to the lectin band on the nitrocellulose strip can be prevented by specific competing sugars. The method can be applied to screen for the presence of lectins in natural materials and to monitor lectin fractions during purification.

A central question in T-cell immunity concerns the nature of intracellular signaling from the antigen receptor, the CD4/CD8 co-receptors and the CD28 antigen. Since the original discovery that T-cell receptors such as CD4 can interact with intracellular protein-tyrosine kinases such as p56lck, remarkable progress has been made in deciphering the signaling pathways that control T-cell growth and immune function. Here, Christopher Rudd and colleagues examine the role of protein-tyrosine kinases, SH2/SH3 domains and lipid kinases in the generation of signals from the TCR zeta/CD3 complex and the CD28 antigen.

Ethanolic extract of A. baccifera was tested for its antiurolithic activity in male albino rats. Urinary stones were induced by implantation of zinc discs in the urinary bladder. The stones formed were mainly of magnesium ammonium phosphate with traces of calcium oxalate. Ethanolic extract of A. baccifera (2g/kg/day, po) was found to be effective in reducing the formation of stones as also in dissolving the pre-formed ones. There was a significant increase in the urinary excretion of calcium, magnesium and oxalate, four weeks after implantation of zinc discs. Treatment with A. baccifera has significantly reduced calcium and magnesium levels in the prophylactic group while it has reversed the levels of these ions to normal values in the curative group.

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cell proliferation. T-cell receptor zeta/CD3 engagement without CD28 ligation leads to a state of nonresponsiveness/anergy, thereby implicating CD28 in the control of peripheral tolerance to foreign antigens or tumors. A key unresolved question has concerned the mechanism by which CD28 generates intracellular signals. Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase with Src-homology 2 (SH2) domain(s) that binds to the platelet-derived growth factor receptor (PDGF-R), an interaction that is essential for signaling by growth factor. In this study, we demonstrate that CD28 binds to PI 3-kinase by means of a Y(P)MXM motif within its cytoplasmic tail. CD28-associated PI 3-kinase was detected by lipid kinase and HPLC analysis as well as by reconstitution experiments with baculoviral-expressed p85 subunit of PI 3-kinase. CD28 bound directly to the p85 subunit without the need for the associated p110 subunit. Site-directed mutagenesis and peptide competition analysis using Y(P)-MXM-containing peptides showed that PI 3-kinase bound to a Y(P)MXM motif within the CD28 cytoplasmic tail (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss of binding, while mutation of M194 caused partial loss of binding. Binding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C- and N-terminal SH2 domains with an affinity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of signaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-kinase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.

Src homology 3 (SH3) domains have been recently shown to bind to proline-rich sequences contained in 3BP1, 3BP2, and SOS. In a recent study we demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) associates with the Fyn SH3 domain. Here we show that p85, the regulatory subunit of PI 3-kinase, binds directly to the SH3 domains of Abl, Lck, Fyn, and p85 itself. An examination of p85 amino acid sequence revealed two proline-rich sequences in its N-terminal region similar to those present in 3BP1, 3BP2, and SOS. To test whether these sequences mediate the association of p85 with SH3 domains two peptides with amino acid composition corresponding to the p85 alpha proline-rich sequences were synthesized and used in competition assays. Both peptides worked equally well in inhibiting the binding of PI 3-kinase activity and p85 alpha to Fyn SH3 domain, whereas a control peptide had no effect. These results indicate that, as in 3BP1 and SOS, the proline-rich sequences in p85 mediate its interaction with SH3 domains. These results also suggest that the SH3 domain of p85 may "self-associate" with the proline-rich motifs of the same subunit as part of the PI 3-kinase regulatory mechanism.

We report a case of poorly differentiated carcinoma of the prostate with lymph node metastasis in a 19-year-old male. He was managed with bilateral orchiectomy, flutamide and recombinant interferon alfa-2b therapy. He showed subjective improvement for 2 months. However, during the 3rd month of treatment there was global deterioration which progressed further till his demise during the 4th month. The beneficial role, if any, of combined hormonal and immunotherapy in advanced prostatic carcinoma is discussed as also the natural history and prognosis of prostatic carcinoma in men under 20 years of age.

The cell surface receptor for IgG Fc domain (Fc gamma RIIIA, CD16) is present as a multimeric complex on human NK cells and is essential for antibody-dependent cellular cytotoxicity. Although CD16 engagement induces tyrosine phosphorylation of several substrates, the intracytoplasmic domains of the CD16 molecule lack intrinsic protein tyrosine kinase consensus sequences. We report here that, in human NK cells, engagement of the CD16 receptor induces tyrosine phosphorylation of p56lck tyrosine kinase. CD16 triggering through anti-receptor antibody enhances in vitro kinase activity of phosphotyrosyl proteins and autophosphorylation activity of anti-lck immunoprecipitates. Similar results were obtained after stimulation with IL-12. In vivo labeling experiments showed that CD16 cross-linking primarily induces in vivo phosphorylation of p56lck at the tyr-394 autophosphorylation site. Using in vitro kinase assay of CD16 immunoprecipitates from lysates of human NK cells, we demonstrated that a phosphorylated protein of 62 to 65 kDa was co-precipitated with CD16. Reprecipitation experiments also suggested that a small amount of p56lck may interact with this complex. These results indicate that CD16 triggering involves p56lck and that this kinase is therefore involved in the signaling pathways regulating NK cell cytolytic functions. However, additional kinases not yet identified are also likely to play a role in the early signaling events through CD16.

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.

Oral administration of oxybutynin chloride is effective in increasing bladder capacity in patients with neurogenic bladder dysfunction who are practising clean intermittent catheterisation, but it is often associated with systemic side effects. The effect of intravesical instillation of oxybutynin chloride was studied in 14 patients who were practising clean intermittent catheterisation and in whom the maximum cystometric capacity was < 250 ml and/or vesical compliance < 5. A 5-mg tablet of oxybutynin chloride was crushed and suspended in 10 ml of boiled and cooled water and instilled into the bladder after emptying it completely; this was carried out thrice daily either by the patient or by his carer. During follow-up (6-12 months) no local or systemic side effects were observed and patient compliance was excellent in 12 patients, who showed an increase in maximum cystometric capacity and vesical compliance. One patient could not retain the drug intravesically because of reflex detrusor contraction and he was excluded from the study. One patient was lost to follow-up. The results suggest that the intravesical instillation of oxybutynin chloride is safe and effective in the treatment of selected patients with neurogenic bladder dysfunction whose bladder capacity is small and who are practising clean intermittent catheterisation.

CD4 serves as a receptor for MHC class II antigens and as a receptor for the human immunodeficiency virus (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. Although anti-CD4 crosslinking may increase lck activity, the effects of HIV-1 gp120 have been controversial. Activated protein-tyrosine kinases are known to associate with certain intracellular proteins possessing src-homology regions (SH-2 domains) such as phosphatidylinositol 3-kinase (PI 3-kinase). In this paper, we demonstrate that the CD4:p56lck complex associates with significant amounts of phosphatidylinositol (PI) kinase activity. High pressure liquid chromatographic (HPLC) analysis of the reaction products demonstrated the presence of phosphatidylinositol 3-phosphate (PI 3-P) and phosphatidylinositol 4-phosphate (PI 4-P), thus indicating that PI 3 and PI 4 kinases associate with CD4-p56lck. The p85 subunit of PI 3-kinase was also detected in anti-CD4 immunoprecipitates by immunoblotting with anti-p85 antiserum. Significantly, p56lck binding to CD4 appears to be necessary for the detection of lipid kinase activity associated with p56lck. Also, anti-HIV gp120 and anti-CD4 crosslinking induced a 10-15-fold increase in levels of both PI 3- and PI 4-kinase activity in anti-CD4 precipitates. Stimulation of CD4-p56lck-linked PI kinases by crosslinked HIV-1 gp120 may play a role in HIV-1-induced immune defects.

The fresh juice of Musa stem (Puttubale) was tested for its antilithiatic activity. Zinc discs were implanted in the urinary bladder of albino rats to induce urolithiasis. The stones formed were mainly of magnesium ammonium phosphate with traces of calcium oxalate. Musa stem juice (3 mL/rat/day orally) was found to be effective in reducing the formation and also in dissolving the pre-formed stones.

The CD4-p56lck and CD8-p56lck complexes have served as a paradym for an expanding number of interactions between src-family members (p56lck, p59fyn, p56lyn, p55blk) and surface receptors. These interactions implicate src-related kinases in the regulation of a variety of intracellular events, from lymphokine production and cytotoxicity to the expression of specific nuclear binding proteins. Different molecular mechanisms appear to have evolved to facilitate the receptor-kinase interactions, including the use of N-terminal regions, SH2 regions and kinase domains. Variation exists in stoichiometry, affinity and the nature of signals generated by these complexes in cells. The CD4-p56lck complex differs from receptor-tyrosine kinases in a number of important ways, including mechanisms of kinase domain regulation and recruitment of substrates such as PI 3-kinase. Furthermore, they may have a special affinity for receptor-substrates such as the TcR zeta, MB1/B29 or CD5 receptors, and act to recruit other SH2-carrying proteins, such as ZAP-70 to the receptor complexes. Receptor-src kinase interactions represent the first step in a cascade of intracellular events within the protein-tyrosine kinase/phosphatase cascade.

The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex.

T cell activation appears to be regulated by an interplay between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). p56lck and p59fyn have been found to associate with CD4 and TCR-CD3 respectively. The CD45 family of transmembrane PTPases has been shown to be able to regulate the activities of these receptor-associated PTKs in vitro. In man, CD45 contains five different isoforms whose distribution defines subsets of T cells having distinct activation requirements and in vitro functions. Several groups have reported a physical interaction between distinct isoforms of CD45 and CD2, CD4, and the TCR-CD3 complex. Given the potential regulatory interaction between CD45 and PTKs in CD4+ subsets expressing different CD45 isoforms, we have examined CD4 associated and TCR-CD3- associated PTK activities, associated phosphatidyl inositol (PI) kinases and substrates of tyrosine phosphorylation in CD45RA+ and CD45RA- CD4+ T cell lines derived from peripheral blood. Both subsets express CD4-associated p56lck and TCR-CD3-associated p59fyn kinases which exhibit identical in vitro phosphorylation at the Y-394 and Y-420 autophosphorylation sites respectively. Further, both subsets exhibited PI kinases activity associated with CD4-p56lck. Consistent with these observations, anti-CD3 crosslinking induced the phosphorylation of a similar spectrum of intracellular substrates in these CD45RA+ and CD45RA- CD4+ T cell lines. These observations indicate that despite the possible interaction between CD45 isoforms and CD4 or TCR-CD3, the mere expression of the CD45RA isoform does not in and of itself alter the presence of receptor-associated kinases or their intracellular targets.

The effect of aluminium (Al) on the supercoiled state of pUC18 DNA was studied by ethidium bromide fluorescence and agarose gel electrophoresis. Al at physiologically relevant concentrations relaxed the intact supercoiled DNA as well as the topoisomers induced by chloroquine. EDTA prevented the unwinding effect of Al on supercoiled DNA. Al did not alter the mobility of linear DNA in agarose gels. The implications of this finding in neurological disorders are discussed.

The CD4 and CD8 antigens on T cells have been shown to associate with the Src family member p56lck and a GTP-binding protein, p32. The identification of receptor interactions with intracellular mediators is essential in the elucidation of downstream signals mediated by engagement of these receptor complexes. In this study, we report the detection of an additional 110-kDa polypeptide (p110) associated with the CD4-p56lck complex in human peripheral blood T lymphocytes and leukemic T-cell lines. p110 bound preferentially to CD4-p56lck as an assembled complex and poorly, if at all, to the individual components. p110 was recognized directly by an antiserum to the C-terminal region of the serine/threonine kinase Raf-1 and is related to a p110 polypeptide detected in anti-Raf-1 immunoprecipitates. Despite its association with the CD4-p56lck complex, p110 was found to be phosphorylated predominantly on serine residues. Furthermore, phorbol ester treatment of cells resulted in a transient increase in the detection of p110 associated with CD4-p56lck, concomitant with the modulation of CD4-p56lck from the cell surface. This Raf-1-related p110 is therefore likely to play a role in signals generated from the CD4-p56lck complex. p110 may serve as a bridge between the CD4-p56lck complex and the serine/threonine kinase pathways of T-cell activation.

T-cell antigens including CD2, CD4, CD6, CD8, and CD28 serve as coreceptors with the T-cell receptor (TCR)/CD3 complex in control of T-cell growth. The molecular basis by which these antigens fulfill this role has remained a major issue. An initial clue to this question came with our finding that the sensitivity of in vitro kinase labeling (specifically using protein-tyrosine kinase p56lck) allowed detection of a physical association between CD4-p56lck and the TCR/CD3 complexes. Another T-cell antigen, CD5, is structurally related to the macrophage scavenger receptor family and, as such, can directly stimulate and/or potentiate T-cell proliferation. In this study, we reveal that in Brij 96-based cell lysates, anti-CD5 antibodies coprecipitated TCR zeta chain (TCR zeta)/CD3 subunits as well as the protein-tyrosine kinases p56lck and p59fyn. Conversely, anti-CD3 antibody coprecipitated CD5, p56lck, and p59fyn. Indeed, anti-CD5 and anti-CD3 gel patterns were virtually identical, except for a difference in relative intensity of polypeptides. Anti-CD4 coprecipitated p56lck, p32, and CD3/TCR zeta subunits but precipitated less CD5, suggesting the existence of CD4-TCR zeta/CD3 complexes distinct from the CD5-TCR zeta/CD3 complexes. Consistent with the formation of a multimeric CD5-TCR zeta/CD3 complex, anti-CD5 crosslinking induced tyrosine phosphorylation of numerous T-cell substrates, similar to those phosphorylated by TCR zeta/CD3 ligation. Significantly, as for TCR zeta, CD5 was found to act as a tyrosine kinase substrate induced by TCR/CD3 ligation. The kinetics of phosphorylation of CD5 (t1/2 = 20 sec) was among the earliest of activation events, more rapid than seen for TCR zeta (t1/2 = 1 min). CD5 represents a likely TCR/CD3-associated substrate for protein-tyrosine kinases (p56lck or p59fyn) and an alternative signaling pathway within a multimeric TCR complex.

We have shown previously that there is a good correlation between the degree of microtubule disassembly by methylmercury (MeHg) and the extent of inhibition of DNA replication in Concanavalin A (Con A)-stimulated mouse splenic lymphocytes. The purpose of this study was to determine if these two events are causally related and to examine the effects of MeHg-induced microtubule disassembly on earlier events of the stimulation process. We show that early steps constituting the activation pathway, such as the Con A-induced increase in Ca2+ influx and the expression of interleukin 2 receptor, are not inhibited by concentrations of MeHg that disassemble microtubules. RNA synthesis is not affected by short-term (3 h) treatment with MeHg, but longer treatment (24 h) inhibits RNA synthesis. In contrast, DNA synthesis is effectively inhibited by a 3-h treatment with MeHg. In lymphocytes treated with taxol, microtubules are not disassembled by MeHg; however, the inhibition of RNA and DNA synthesis persists. We conclude that the inhibition of nucleic acid synthesis by MeHg is not causally related to MeHg-induced microtubule disassembly.

The effect of the lipophilic gold compound, auranofin (AUR) on the calcium homeostasis of human neutrophils treated with or without n-formyl-methionyl-leucyl-phenylalanine (FMLP) was investigated. In agreement with previous reports, FMLP induced a rapid release of intracellular Ca2+ stores followed by a smaller influx of extracellular Ca2+. AUR and staurosporine enhanced while phorbol 12-myristate 13-acetate suppressed the secondary influx of Ca2+. Mn2(+)-quenching-of-fluorescence studies indicate that phorbol 12-myristate 13-acetate incubation blocked cation entry. AUR or staurosporine potentiation of FMLP effects on cytoplasmic free Ca2+ [( Ca2+]i) was attributed to suppression of negative feedback effects of protein kinase C. AUR (5-45 microM) per se induced a slow release of internal Ca2+ stores followed by a delayed influx of extracellular Ca2+. Control studies showed that AUR did not induce the formation of inositol 1,4,5-trisphosphate, lyse cells, or promote dye leakage. Dithiothreitol suppressed the AUR effect. AUR triggered biphasic but smaller increases in [Ca2+]i of neutrophil cytoplasts. Studies with permeabilized neutrophils showed that AUR directly released Ca2+ from internal stores. By comparison, gold sodium thiomalate, which had no effect on intact cells, also released Ca2+ from permeabilized cells. Present results indicate that AUR modulated [Ca2+]i directly by mobilized Ca2+ from multiple storage sites and indirectly by inhibiting protein kinase C.

During the differentiation of the clonally distributed lymphocytes of mouse and man into mature resting B and T cells, their DNA becomes tightly packed into dense heterochromatin masses and exhibits very little transcriptional activity; it also becomes extensively nicked, containing some 3000-4000 single-strand breaks per diploid genome. The nuclear matrix is sparse and poorly organized and there are but trace amounts of the matrix-linked enzyme DNA topoisomerase II; the nucleus of these small cells is surrounded by a thin rim of cytoplasm. The resting cell can thus be considered (by analogy to a sperm cell) as a vector for transporting tightly packed and relatively inert genetic information to all parts of the body. When the lymphocyte is stimulated to enter a proliferative cycle by binding of appropriately presented antigen or mitogen to relevant membrane receptors, the cell enlarges, due to increased synthesis of protein; the dense heterochromatin is pulled out into very small clumps, as a result of an enormous growth in size as well as complexity of the nuclear matrix, and a great increase in transcriptional activity occurs. We have identified four nuclear matrix antigens that are very widely conserved in the evolution of eucaryotes and that occupy distinctive domains in interphase nuclei. Of particular interest is antigen P1, detected in organisms ranging from algae to mammals. By virtue of its location at the interface between nuclear envelope and chromatin, we propose that it plays a major and evolutionarily conserved role in chromatin organization and orientation in all eucaryotic cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

In view of our previous studies that the plasma elimination of norethisterone (NET) from mini pill is faster in low socio-economic group Indian women, the present studies were contemplated to find the least effective dosage of NET from combination pills. Pharmacokinetics of NET were evaluated in a total of twenty women of low socio-economic group taking pills containing NET-acetate (500 micrograms or 1 mg) and ethinyl estradiol (30 or 50 micrograms respectively) on empty stomach. Blood samples were drawn at different time intervals from 0.5 to 24 hr and plasma NET was estimated by a specific radio-immunoassay. In the women taking 1 mg NET-acetate containing pills peak plasma levels ranging from 6.2 to 20.8 ng/ml were observed at 1 hr whereas with 500 micrograms pill they ranged from 2.0 to 6.5 ng/ml and the peak was noted at 4 hr. Pharmacokinetic parameters of NET were more or less comparable between the two pills. The results suggest that pills containing 500 micrograms NET-acetate and 30 micrograms ethinyl estradiol provide adequate levels of NET even in low-socio-economic group women.

Plasma norethindrone (NET) and progesterone were estimated by radioimmunoassay in seven Indian women after intramuscular administration of 20 mg NET enanthate. One subject had intermenstrual bleeding throughout the cycle. Out of the six subjects considered for analysis, three subjects showed ovulation suppression, two had delayed ovulation and the remaining one exhibited normal ovulatory pattern. Post-peak average plasma NET values ranged from 1.0 to 2.1 ng/ml. These values showed a significant positive correlation with the anthropometric indices such as body weight and mid-arm-circumference. The subjects with lower anthropometry showed exponential decline of plasma NET. A possible role for nutritional status of an individual in drug disposition is indicated from this study.

The pharmacokinetics of norethindrone (NET) were studied in adult female rabbits and rhesus monkeys with a view to developing an animal model closely resembling humans in the handling of NET. Plasma levels of NET were determined in rabbits and monkeys after administering either labelled NET or a NET minipill. Pharmacokinetic parameters were evaluated by using a two-compartment open model and by graphical and regression analysis of plasma NET data. In rabbits, the drug absorption was found to be rapid and NET peak levels were attained within 0.5 to 1.0 hour. The clearance half-lives were 1.3 hours for 'alpha' and 10.0 hours for 'beta'. In monkeys, plasma NET values showed an inconsistent pattern and the approximate t 1/2 was found to lie between 4 and 6 hours. The observed pharmacokinetics of NET in rabbits rather than in monkeys were closer to those in humans. On this basis, it would appear that the rabbit is a suitable animal model for studying effects of nutritional factors on the pharmacokinetics of NET.

The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 X 10(3) +/- 12 X 10(3)/cell in resting to 60 X 10(3) +/- 27 X 10(3)/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: there was an increase in the Vmax of ouabain binding, without variation in the Km; the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane.

Two early events in the mitogen-induced entry of murine splenocytes into proliferation are (a) a rapid rise in influx of Na+, causing its total internal concentration to increase by 42 +/- 7% within 2 h of culture with concanavalin A (Con A), and (b) rejoining of some 3000 DNA strand breaks per diploid genome within the same period. Con A did not induce rejoining in low Na+ (less than 9 mM) medium, but the process began directly when Na+ was added, at its usual concentration, to the growth medium. Incubation of cells with ouabain, an inhibitor of the Na+K+-ATPase, or monensin, a Na+ ionophore, caused an increase in the internal Na+ concentration in normal, but not in low, Na+ medium. In the former (but not in the latter) medium, both ouabain and monensin caused rejoining of the DNA strand breaks to occur in resting lymphocytes, i.e., in the absence of mitogen. Stimulation of splenocytes with Con A also resulted in a rapid but transient increase in the level of intracellular free Ca2+. This effect was also observed in the absence of extracellular Na+; however, deprivation of extracellular Ca2+ completely abolished this effect. Moreover, the intracellular free Ca2+ level was significantly higher in cells suspended in Na+-free buffer or medium. Since the Con A-induced rejoining of DNA strand breaks occurred in the absence of extracellular Ca2+ and removal of extracellular Na+ had no inhibitory effect on the Con A-induced increase in the level of intracellular free Ca2+, the Con A-stimulated repair could not have been mediated by the initial increase in Ca2+ influx. The early mitogen-induced increase in the internal Na+ concentration is a necessary and sufficient signal for the rejoining of breaks, an event that must occur before the proliferating lymphocytes can replicate their DNA.

Stimulation of pig peripheral blood lymphocytes with concanavalin A (Con A) provoked a rapid increase (two- to threefold) in the rate of ouabain-inhibitable K+ uptake observable within 3-10 min of stimulation with mitogen. At least two phases can be distinguished in the activation of the Na+/K+ pump: the early phase (till 3 h) is characterized by an unaltered number of ouabain binding sites and the later phase (noted at 5 h) by an increased number of such sites. Both K+ efflux and influx increased to the same extent, thereby maintaining [K+]i at the same level as in resting cells (120 mM). Within 3 min of addition of mitogen, the rates of total and amiloride-inhibitable Na+ uptake went up two- and fourfold, respectively, thus resulting in rapid increase in [Na+]i from 20 to about 50 mM. Activation of the Na+/K+ pump was not observed when the cells were stimulated with Con A in low Na+ medium (9 mM), nor did the usual rise in [Na+]i occur. When monensin (30 microM), a Na+/H+ ionophore, was added to resting cells, an increase in both [Na+]i and active K+ uptake occurred in normal medium but not when cells were suspended in low Na+ isotonic buffer. Amiloride (500 microM), on the other hand, prevented both the Con A-induced increase in [Na+]i and the activation of the Na+/K+ pump. Despite complete inhibition of the Na+,K+-ATPase in the presence of ouabain (1 mM), Con A activated the amiloride-inhibitable Na+ uptake in the usual way. In mouse splenocytes stimulated with Con A, there was also a parallel rise in both [Na+]i and active K+ uptake but this took considerably longer to occur than was the case in pig peripheral blood lymphocytes. Increase in both ionic fluxes, the former passive and the latter active, is essential to the entry and maintenance of the cells in proliferative cycle.

In this paper we explore the applicability of the soft mode approach to study the conformational transitions of DNA. It is believed that the A-B conformation change is a first order transition. Soft mode theories only apply to the initial stages of a first order transition. However the mode softening in such a case can be the initiating factor which ultimately leads to the transition. The first order transition is, then, a breakdown of what otherwise would have been a true second order transition. The mode softening is causally connected to onset of the transition. We use the eigenvectors obtained from lattice dynamics calculations to identify the softmode. We use the eigenvector projections to form a force constant matrix that is required to drive a mode soft. We explore the methods by which this force constant matrix can be formed. We suggest that the breaking of specific "water bridges" between phosphate groups in the two single strands can drive the conformation change.

Our previous studies have shown that the plasma norethindrone (NET) elimination is faster in women of low socio-economic group as compared to those of high socio-economic group. Undernutrition being very common in low socio-economic group, using an animal model, efforts were made to determine whether these differences were due to differences in the nutritional status of these two group of women. The experimental animals consisted of a set of twelve female rabbits, divided equally into two groups. One group (A) was fed on a stock diet ad libitum while the second group (B) received the diet in restricted amounts (half of that consumed by animals of group A). At the end of three months, radiolabelled NET was administered intravenously and plasma drug levels were determined at frequent intervals up to 24 hours. The plasma values were subjected to pharmacokinetic analysis. Food restriction to the extent of 50% resulted in a 20% deficit of body weight as compared to controls. The half-lives of alpha and beta phases were shortened in the restricted group resulting in an enhanced metabolic clearance rate of NET in this group. Though the differences were not statistically significant, the volume of distribution tended to be lower and the dose excreted in urine higher when food restriction was imposed.

The calculated phonon spectrum of Z-form poly(dG-dC).poly(dG-dC) between 400 and 1600 cm-1 is reported. Comparison with the available data shows the very good agreement between theory and experiment. The eigenvector displacement is used to assign the characteristics of some of the important modes.

The present investigation was designed to understand the mechanism underlying the altered pharmacokinetic and pharmacodynamic properties of norethindrone and levonorgestrel observed in undernourished rabbits. Eighteen albino rabbits aged 3-4 months were divided into two groups. One group received a stock diet ad libitum and the other group was individually fed half the food consumed by the matched animal in ad lib group. Three pairs of animals were sacrificed at the end of three months and the remaining at the end of five months. Liver and uterine tissues were dissected out and processed for analysis. For both sets of animals, whether killed at the end of three months or 5 months, the trend of results obtained were similar. The capacity and affinity characteristics of plasma sex hormone binding globulin were found to be not significantly different between control and experimental animals. The specific activities of liver microsomal glucuronyl transferase and cytochrome P-450 were significantly higher in undernourished rabbits. The increase observed in the activity of sulfuryl transferase was, however, not statistically significant. Uterine cytosol progesterone receptor levels were found to be higher in food restricted animals though there were no changes in the affinity properties of the receptors. Thus, it appears that imposition of food restriction leads to increased drug metabolism as a result of increased drug metabolizing enzyme activities and that the animals appear to adapt by enhanced target organ response in terms of elevated receptor concentration.

Pharmacokinetics of norethindrone (NET) was evaluated in eleven women belonging to a low socio-economic group and in five womem belonging to the high socio-economic group after the administration of an oral dose of 0.35 mg NET minipill on an empty stomach. Blood samples were collected at different intervals of time over a period of 24 hours. Plasma NET was estimated by radioimmunoassay. In all women, peak levels of NET occurred within 1-2 hours and a semi-log plot of plasma NET levels showed a biexponential decline. The half-life of plasma NET clearance was relatively shorter in women of low socio-economic group with poor nutritional status as indicated by anthropometric indices, as compared to that in well nourished women of high socio-economic group. There was a significant positive correlation between weight/(height) 2x 100 index on the one hand and t1/2 (beta) on the other in all the women studied, thereby suggesting a role for nutritional status in the metabolic handling of NET.

Mild-moderate anemia (hemoglobin, 10-12 g/100 ml) was induced in eight female rabbits by feeding a purified diet with low iron content over a period of 4 months and it could be maintained for a further period of 7 months. Nine control animals received the same diet supplemented with iron. Pharmacokinetics of intravenously administered contraceptive steriods, norethindrone and levonorgestrel, were determined in both control and anemic rabbits at the end of 150 and 171 days, respectively. No significant alterations were observed in the disposition of these steroids in deficient rabbits as compared to controls, indicating that mild-moderate forms of iron deficiency anemia may not influence the metabolic handling of either norethindrone or levonogestrel.