Analysis of mutational specificity induced by heterocyclic amines in the lacZ gene of Escherichia coli

Mutational spectra induced by different heterocyclic amines were characterized and compared with those obtained from diethylnitrosamine and N-methyl-N-nitrosourea. Mutation classes were identified by means of a series of mutant lacZ genes in F' episomes in Escherichia coli engineered to detect specifically each of two transitions, four transversions and five kinds of frameshift events. More than 99.5% of the mutations induced by heterocyclic amines were frameshift mutations. -2(C.G-G.C) frameshifts were favored over other types, such as +1(G.C), -1(G.C), +1(A.T) and -1(A.T), except when 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was administered. -1(G.C) and +1(G.C) frameshifts predominate following Trp-P-1 treatment. A small number of G.C-->T.A transversions were induced by the treatment with 2-amino-3,4-dimethylimidazo[4,5-f]quinoline as well as with several other heterocyclic amines examined. Since G.C-->T.A transversions, but not frameshift mutations, are reported to play a role in heterocyclic amine-induced activation of the c-Ha-ras protooncogene or inactivation of the p53 tumor suppressor gene, the low level of base substitutions, particularly G.C-->T.A transversions, may represent a partial explanation for the relatively modest carcinogenic activity of heterocyclic amines, despite their extraordinarily strong mutagenicity in the Salmonella mutation assay.     

Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis

The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or [Leu19]-recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser19 and Ser40 in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in L-DOPA biosynthesis rates in intact AtT-20 cells. Treatment of either PC12 cells or AtT-20 cells expressing wild-type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser19 (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser40 phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser40, as opposed to Ser19, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the increases in the phosphorylation state of Ser40 but not Ser19. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT-20 cells expressing rTH in which Ser19 phosphorylation had been eliminated (by substitution of Leu for Ser19). Thus, depolarization-induced increases in the stoichiometry of Ser19 phosphorylation do not appear to influence directly the activity of TH in situ.     

Synergistic enhancement of small and large intestinal carcinogenesis by combined treatment of rats with five heterocyclic amines in a medium-term multi-organ bioassay

The carcinogenic potential of five heterocyclic amines in combination was analyzed using a medium-term multi-organ bioassay. Male F344 rats were initially treated with five known carcinogens (diethylnitrosamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl)-nitrosamine, 1,2-dimethylhydrazine and 2,2'-dihydroxy-di-n-propylnitrosamine) over a 4 week period to induce preneoplastic changes in a variety of organs (wide spectrum initiation) and then given the five heterocyclic amines, all having the intestines as a target of their carcinogenicity, individually or in combination in the diet for a further 24 weeks. In the small and large intestines, simultaneous administration of five heterocyclic amines at doses 1/5 or 1/25 of those used in reported carcinogenicity studies resulted in higher incidences and multiplicities of adenocarcinomas than expected from the five individual effects, although the differences were not statistically significant. A synergistic effect based on the additive model was most evident (P < 0.141) with multiplicity data for carcinoma in the small intestine at the 1/25 dose. A similar trend was observed for Zymbal gland (P < 0.077), but not other carcinoma induction. Thus the results suggested that synergism depends on the carcinogenic organotropism of individual agents as well as the doses applied in combination.     

Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson's disease

To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.     

In vivo phosphorylation of the epithelial sodium channel

The activity of the epithelial sodium channel (ENaC) in the distal nephron is regulated by an antidiuretic hormone, aldosterone, and insulin, but the molecular mechanisms that mediate these hormonal effects are mostly unknown. We have investigated whether aldosterone, insulin, or activation of protein kinases has an effect on the phosphorylation of the channel. Experiments were performed in an epithelial cell line generated by stable cotransfection of the three subunits (alpha, beta, and gamma) of ENaC. We found that beta and gamma, but not the alpha subunit, are phosphorylated in the basal state. Aldosterone, insulin, and protein kinases A and C increased phosphorylation of the beta and gamma subunits in their carboxyl termini, but none of these agents induced de novo phosphorylation of alpha subunits. Serines and threonines but not tyrosines were found to be phosphorylated. The results suggest that aldosterone, insulin, and protein kinases A and C modulate the activity of ENaC by phosphorylation of the carboxyl termini of the beta and gamma subunits.     

CREB activation mediates plasticity in cultured hippocampal neurons

Activation of cyclic AMP dependent kinase is believed to mediate slow onset, long-term potentiation (LTP) in central neurons. Cyclic-AMP activates a cascade of molecular events leading to phosphorylation of the nuclear cAMP responsive element binding protein (pCREB). Whereas a variety of stimuli lead to activation of CREB, the molecular processes downstream of CREB, which may be relevant to neuronal plasticity, are yet largely unknown. We have recently found that following exposure to estradiol, pCREB mediates the large increase in dendritic spine density in cultured rat hippocampal neurons. We now extend these observations to include other stimuli, such as bicuculline, that cause the formation of new dendritic spines. Such stimuli share with estradiol the same mechanism of action in that both require activity-dependent CREB phosphorylation. Our observations suggest that CREB phosphorylation is a necessary, but perhaps not sufficient, step in the process leading to the generation of new dendritic spines and perhaps to functional plasticity as well.     

Insulin-induced vasodilation is dependent on tetrahydrobiopterin synthesis

Insulin has been shown to elicit vasodilation through increases in nitric oxide (NO) production. To examine whether insulin may modulate the availability of tetrahydrobiopterin (BH4) (an absolute cofactor requirement for NO synthase activation), we studied the effects of insulin (150 nmol/L) on femoral arterial reactivity (to norepinephrine [NE]) in the presence and absence of 2,4-diamino-6-hydroxypyrimidine (DAHP), a specific inhibitor of BH4 production. Our data indicate that inhibition of BH4 synthesis results in an attenuation in the vasodepressor effect of insulin. One possibility is that insulin may regulate NO production by increasing cofactor (BH4) availability for activation of NO synthase.     

Interaction of Janus kinases JAK-1 and JAK-2 with the insulin receptor and the insulin-like growth factor-1 receptor

Insulin and insulin-like growth factor-1 (IGF-1) treatment of cells overexpressing the insulin receptor or the IGF-1 receptor promotes phosphorylation and activation of Janus kinases JAK-1 and JAK-2 but not of TYK-2. With insulin, we observed maximal phosphorylation of JAK-1 within 2 min (5.2 +/- 0.6-fold) and maximal phosphorylation of JAK-2 within 10 min (2.4 +/- 0.6-fold). In cells incubated with IGF-1, we found maximal phosphorylation of JAK-2 within 2 min (1.9 +/- 0.2-fold) and of JAK-1 within 5 min (4.5 +/- 0.4-fold). The JAKs from insulin- or IGF-1-stimulated cells were activated, as shown by their autophosphorylation in vitro. Moreover, they were able to phosphorylate in vitro native insulin receptor substrate (IRS)-1 and a fragment of IRS-2 (GST-IRS-2591-786). Comparison of 32P-peptide maps of IRS-1 phosphorylated in vitro by the insulin receptor vs. JAK-1 showed the occurrence of different phosphopeptides, suggesting that different sites are likely to be phosphorylated by the two kinases. Finally, coprecipitation of receptors and JAK-1 was seen, and phosphorylation of both receptors was found to be necessary for receptor binding to JAK-1. Two domains of JAK- 1 are involved in the formation of the complex between receptor and JAK-1, i.e. the N-terminal portion containing JH7 and JH6 domains, and the C-terminal kinase domain (JH1 domain). Taking our data together, we conclude that: 1) insulin and IGF-1 lead to phosphorylation and activation of JAK-1 and JAK-2 in intact cells; 2) phosphorylation of IRS-I by JAK-1 seems to occur on sites different from those phosphorylated by the insulin receptor; 3) JAK-1 interacts directly with phosphorylated insulin and IGF-1 receptors; and 4) the JH7-JH6 and JH1 domains of JAK-1 are responsible for the interaction with insulin and IGF-1 receptors.     

Low levels of ionic mercury modulate protein tyrosine phosphorylation in lymphocytes

The ability of ionic mercury to induce protein tyrosine phosphorylation in mouse spleen cells and in the mouse WEHI-231 B-cell lymphoma was investigated. We have confirmed previous studies which showed that exposure to high levels (several hundred microM) of mercury lead to very large increases in the level of protein tyrosine phosphorylation in these cell systems. However we have also demonstrated that low levels (in the order of 0.1 to 1.0 microM) of mercury also significantly upregulate protein tyrosine phosphorylation. Mercury induced protein tyrosine phosphorylation is inhibited by the mercury chelator penicillamine and by pretreating treating target cells with the sulfhydryl blocking reagent N-hydroxymaleimide. These results suggest that exposure to low levels of mercury could potentially interfere with lymphocyte signal transduction and so offer a possible explanation as to how mercury exposure could lead to immune cellular dysfunction. On a molecular level, the results suggest that the site(s) of action with respect to mercury dependent induction of protein tyrosine phosphorylation is likely a free disulfide group or groups located on the outer leaflet of the plasma membrane.     

Gender differences in DSM-IV alcohol use disorders and major depression as distributed in the general population: clinical implications

This study examined gender differences within and between five groups of subjects drawn from a large representative sample of the United States population and classified as having either major depression (MDD) only, alcohol use disorder (AUD) only, or primary, secondary, or concurrent depression to determine if these diagnostic profiles (1) were consistent with those drawn on clinical samples and (2) might suggest potential clinical implications. Respondents (N = 9,985) from a nationally representative survey of the United States population met DSM-IV criteria for classification into these five mutually exclusive groups that were compared within and between groups by gender on the characteristics of each disorder. The results were consistent with those of other studies: (1) gender distributions of AUD and depressive disorder remain almost mirror opposites, and (2) comorbid disorders are more severe than either of the conditions appearing singly. Findings of particular interest were that the synergistic effects of an alcohol and a depressive condition operate equally for both men and women with concurrent depression. This points to the necessity of attending carefully to gender biases when dealing with comorbid conditions, last we fail to take alcoholism in the presence of depression seriously enough in women and vice versa in men. Additionally, women with primary depression are at high risk for suicide and thus may require special attention in the evaluative phase of treatment.     

Cys860 in the extracellular domain of insulin receptor beta-subunit is critical for internalization and signal transduction

The C860S mutation (IRC860S) in the extracellular domain of the insulin receptor beta-subunit has previously been shown to result in an inhibition of insulin receptor internalization. The present work aims at further dissecting the consequences of this mutation not only on insulin receptor internalization, but also on the signaling of the receptor. Following transfection of Chinese hamster ovary (CHO) cells with insulin receptors with the C860S mutation (CHO-IRC860S) and quantitative electron microscopic analysis of [125I]insulin localization in these cells, the inhibition of receptor internalization appears to be due to an inhibition of the lateral translocation of the receptor from microvilli to nonvillous domains of the cell surface. At 37 C, insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation is inhibited by 50% in CHO-IRC860S, whereas Shc phosphorylation remains unaffected. The inhibition of IRS-1 phosphorylation is still present when experiments are conducted at 4 C, a temperature at which insulin receptor internalization is prevented, suggesting that the defect in IRS-1 phosphorylation is not due to the reduced internalization of the receptor. In terms of biological effects, the mutation has negative consequences on insulin-stimulated c-fos expression and DNA synthesis as well as on glycogen synthase activity. Eventually, the events observed are specific for Cys860, as individual substitution of the two more proximal Cys residues (795 and 872) to Ser is not accompanied by any change in either insulin-induced insulin receptor internalization or IRS-1 phosphorylation. Thus, the present analysis of CHO-IRC860S 1) reveals that insulin receptor surface redistribution is not solely dependent on receptor autophosphorylation, 2) emphasizes that IRS-1 phosphorylation is not dependent on receptor internalization and can be triggered from microvilli, and 3) stresses divergent aspects between two of the major signaling pathways of the insulin receptor.     

The pleckstrin homology and phosphotyrosine binding domains of insulin receptor substrate 1 mediate inhibition of apoptosis by insulin

Insulin and insulin-like growth factor 1 (IGF-1) evoke diverse biological effects through receptor-mediated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. We investigated the elements of IRS-1 signaling that inhibit apoptosis of interleukin 3 (IL-3)-deprived 32D myeloid progenitor cells. 32D cells have few insulin receptors and no IRS proteins; therefore, insulin failed to inhibit apoptosis during IL-3 withdrawal. Insulin stimulated mitogen-activated protein kinase in 32D cells expressing insulin receptors (32DIR) but failed to activate the phosphatidylinositol 3 (PI 3)-kinase cascade or to inhibit apoptosis. By contrast, insulin stimulated the PI 3-kinase cascade, inhibited apoptosis, and promoted replication of 32DIR cells expressing IRS-1. As expected, insulin did not stimulate PI 3-kinase in 32DIR cells, which expressed a truncated IRS-1 protein lacking the tail of tyrosine phosphorylation sites. However, this truncated IRS-1 protein, which retained the NH2-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, mediated phosphorylation of PKB/akt, inhibition of apoptosis, and replication of 32DIR cells during insulin stimulation. These results suggest that a phosphotyrosine-independent mechanism mediated by the PH and PTB domains promoted antiapoptotic and growth actions of insulin. Although PI 3-kinase was not activated, its phospholipid products were required, since LY294002 inhibited these responses. Without IRS-1, a chimeric insulin receptor containing a tail of tyrosine phosphorylation sites derived from IRS-1 activated the PI 3-kinase cascade but failed to inhibit apoptosis. Thus, phosphotyrosine-independent IRS-1-linked pathways may be critical for survival and growth of IL-3-deprived 32D cells during insulin stimulation.     

Combination of insulinomimetic agents H2O2 and vanadate enhances insulin receptor mediated tyrosine phosphorylation of IRS-1 leading to IRS-1 association with the phosphatidylinositol 3-kinase

To analyze the mechanism of action of the insulinomimetic agents H2O2, vanadate, and pervanadate (H2O2 and vanadate), CHO cells or CHO cells that overexpress wild-type or mutant insulin receptor and/or the insulin receptor substrate (IRS-1) were used. H2O2 or vanadate treatment alone had little or no effect on tyrosine phosphorylation of cellular proteins; however, pervanadate treatment dramatically enhanced tyrosine phosphorylation of a number of proteins including the insulin receptor and IRS-1. However, the insulin receptor and IRS-1 coimmunoprecipitate from insulin-treated but not from pervanadate-treated cells. Pervanadate-induced tyrosine phosphorylation of the insulin receptor led to an increase in insulin receptor tyrosine kinase activity toward IRS-1 in vivo and IRS-1 peptides in vitro equal to that induced by insulin treatment. Pervanadate-enhanced phosphorylation of IRS-1 led to a fifteenfold increase in IRS-1-associated phosphatidylinositol (PtdIns) 3-kinase activity. However, insulin receptor-associated PtdIns 3-kinase activity from pervanadate-treated cells was not detectable, while insulin receptor-associated PtdIns 3-kinase activity from insulin-treated cells was 20% of the IRS-1-associated activity. Thus, pervanadate but not H2O2 or vanadate alone under these conditions mimics many of insulin actions, but pervanadate treatment does not induce insulin receptor/IRS-1 association.     

A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation

Tumor necrosis factor alpha (TNFalpha) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFalpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFalpha for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFalpha effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFalpha on insulin action.     

Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591-786

We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system. Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively. The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1. Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1. This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation.     

Current concepts of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) may be loosely defined as unexplained hyperandrogenism, with variable degrees of cutaneous symptoms, anovulatory symptoms, and obesity. The vast majority of patients with the full-blown Stein-Leventhal syndrome have functional ovarian hyperandrogenism (FOH). However, FOH often occurs without the LH excess or polycystic ovaries of classic PCOS. Functional adrenal hyperandrogenism (FAH) is often found in the syndrome, but it is less closely associated with anovulatory symptoms than is FOH. The vast majority of FOH seems to arise from abnormal regulation (dysregulation) of ovarian androgen secretion. This typically is due to escape from desensitization to luteinizing hormone (LH); this appears to occur because of a breakdown in the processes that normally coordinate ovarian androgen and oestrogen secretion so as to prevent hyperoestrogenism. Similar dysregulation of adrenal androgen secretion in response to ACTH seems to account for most FAH. Dysregulation of androgen secretion may affect the ovary alone (isolated FOH), the adrenal alone (isolated FAH), or both together. Modest insulin resistance is common in PCOS/FOH, and the resultant hyperinsulinaemia is a major candidate as the cause of the dysregulation. The hyperinsulinaemia may arise from either 'nature' (genetic defects) or 'nurture' (exogenous obesity). Although hyperinsulinaemia alone does not have an obvious effect on steroidogenesis, it may act in genetically predisposed women as a 'second hit' to unmask latent abnormalities in steroidogenesis. The ovary, the adrenal cortex, and several other organs paradoxically function as if responding to the hyperinsulinaemic state in spite of resistance to the effects of insulin on glucose metabolism. PCOS should be viewed as an early manifestation of a hyperinsulinaemic condition that will predispose to cardiovascular and metabolic complications later in life. A subset of PCOS patients appear to have not only insulin resistance but also beta-cell secretory dysfunction, which may indicate a relationship of the disorder to NIDDM. The fundamental genetic defects remain to be elucidated.     

Protein tyrosine kinases couple the surface immunoglobulin of germinal center B cells to phosphatidylinositol-dependent and -independent pathways of rescue from apoptosis

Considerable progress has been made recently in elucidating the intracellular signal transduction pathways which couple surface immunoglobulin (sIg) of resting B lymphocytes (BH) to the proliferative cycle. By contrast, nothing is known of the signals which couple the sIg of germinal center (GC) B cells not to mitogenesis but, instead, to the suppression of apoptosis: the present study examines the signaling pathways through which this response is achieved. GC B cells treated with anti-Ig exhibited enhanced phosphorylation on tyrosine for a number substrates: this was accompanied by a transient increase in inositol 1,4,5-trisphosphate, an increase in [Ca2+]i, and translocation of PKC from the cytosol. These changes could be provoked with Abs specific for IgG or IgA, the major sIg on GC B cells. Herbimycin A, an inhibitor of protein tyrosine kinases (PTK), uncoupled sIg on GC B cells from both the increase in [Ca2+]i and the rescue from apoptosis: the latter was only partially blocked by inhibitors of PKC and chelators of intracellular and extracellular Ca2+. These data indicate that not only do PTK link the antigen receptor (AgR) of GC B cells to both phosphatidylinositol (PI)-dependent and -independent routes of survival but also that tyrosine phosphorylation is critical for sIg-mediated rescue of this population from apoptosis. Moreover, despite the distinct functional responses observed following ligation of the AgR of resting BH lymphocytes and GC B cells, anti-Ig initiates a very similar pattern of second messenger change in these populations suggesting that bifurcation must occur at a more distal stage of the signaling process.     

Zinc, insulin and diabetes

Miso, a widely used Japanese fermented food was analysed for its lactic acid bacterial count on bromocresol purple agar. The binding of eight different foodborne carcinogenic heterocyclic amines to 25 bacterial isolates from miso were investigated. The heterocyclic amines used were 3-amino-1,4-dimethyl[5H]pyrido(4,3-b)indole (Trp-P-1), 3-amino-1-methyl[5H]pyrido(4,3-b)indole (Trp-P-2), 2-amino-6-methyldipyrido(1,2-a:3'2'-d)imidazole (Glu-P-1), 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-dimethylimidazo(4,5f)quinoline (IQ), 2-amino-3,4-dimethylimidazo(4,5-f) quinoline (MeIQ), 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), and 2-amino-3-methyl-9H-pyrido(2,3)indole (MeA alpha C). The lyophilized cells of all of the isolates exhibited high binding activity towards Trp-P-1, Trp-P-2, MeA alpha C, and PhIP, while Glu-P-1 and IQ were not effectively bound. Of the isolates tested, the strongest and weakest binders were identified as Pediococcus acidilactici 1 and 2, respectively. Lyophilized cell wall fractions, heat-treated cells, and the cytoplasmic contents of P. acidilactici 1 and 2 were analysed for their ability to bind to different mutagens. Pure cell wall and peptidoglycan showed greater binding activity than the bacterial cells. Cytoplasmic content also showed some binding, but it was much less effective. The impact of enzymes (amylase, protease, cellulase, chitinase, muraminase, and peptidase) and acetylation of Trp-P-1 and IQ on the binding action of bacteria and cell wall material were also analysed to understand the possible processes involved in the binding of lactic acid bacteria to carcinogenic heterocyclic amines.     

Role of the major histocompatibility complex in T cell activation of B cell subpopulations. A single monoclonal T helper cell population activates different B cell subpopulations by distinct pathways

It has recently been demonstrated that the Lyb-5+ and Lyb-5- B cell subpopulations differ in their requirements for major histocompatibility complex (MHC)-restricted activation by T helper (TH) cells. To determine whether these MHC-restricted and -unrestricted pathways of B cell activation result from differences in the participating TH cell populations or reflect differences exclusively in the responding B cell subpopulations, experiments were carried out using cloned TH cells for in vitro antibody responses to trinitrophenyl-keyhole limpet hemocyanin. The same cloned T helper cells were able to activate both CBA/N (Lyb-5-) B cells and CBA/CaHN (Lyb-5+ + Lyb-5-) B cells under different experimental conditions. The activation of Lyb-5-B cells by cloned T helper cells required both MHC-restricted TH cell-B cell interaction and carrier-hapten linkage. In contrast, the activation of Lyb-5+ B cells required only MHC-restricted T helper cell interaction with accessory cells, while T-B interaction was MHC unrestricted and did not require carrier-hapten linkage. Thus, the differences in activation requirements observed for the Lyb-5- and Lyb-5+ B cell subsets do not result from differences in the TH cell populations activating these B cells, but rather reflect differences in the ability of these B cells to respond to signals from the same TH cells.