Two new male contraceptives exert their effects by depleting germ cells prematurely from the testis

The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3-7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4-14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.     

Phosphorylation of the catalytic subunit of rat renal Na+, K+-ATPase by classical PKC isoforms

In this study we have evaluated the specificity of different PKC isozymes for the phosphorylation of the catalytic alpha1 subunit of rat renal Na+,K+-ATPase (alpha1 Na+,K+-ATPase). Using in vitro phosphotransferase assays we found that classical PKCs (cPKCs) alpha, betaI, and gamma efficiently phosphorylate alpha1 Na+,K+-ATPase. However, alpha1 Na+,K+-ATPase was a poor substrate for the novel PKCs (nPKCs) delta and epsilon. Two-dimensional phosphopeptide mapping revealed a similar pattern of phosphorylation by all cPKCs. The functional significance of this finding was evaluated by measuring Na+,K+-ATPase activity (assessed by 86Rb+ uptake) in COS-7 cells expressing the rat alpha1 Na+,K+-ATPase. 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a nonselective PKC activator, inhibited Na+,K+-ATPase activity in this system. On the other hand, 12-deoxyphorbol-13-phenylacetate (DPP), which preferentially activates nPKCepsilon, did not affect 86Rb+ uptake. These results indicate a differential pattern of phosphorylation and regulation of rat renal Na+,K+-ATPase activity by PKC isoforms and suggest an important role for cPKCs in the physiological regulation of the pump.     

Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant

Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.     

Effect of divalent metal cations on the dimerization of OXA-10 and -14 class D beta-lactamases from Pseudomonas aeruginosa

The factors influencing the oligomerization state of OXA-10 and OXA-14 class D beta-lactamases in solution have been investigated. Both enzymes were found to exist as an equilibrium mixture of a monomer and dimer, with a K(d) close to 40 microM. The dimeric form was stabilized by divalent metal cations. The ability of different metal ions to stabilize the dimer was in the following order: Cd(2+) > Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) > Mn(2+) > Ca(2+) > Mg(2+). The apparent K(d)s describing the binding of Zn(2+) and Cd(2+) cations to the OXA-10 dimer were 7.8 and 5.7 microM, respectively. The metal ions had a profound effect on the thermal stability of the protein complex observed by differential scanning calorimetry. The enzyme showed a sharp transition with a T(m) of 58.7 degrees C in the absence of divalent cations, and an equally sharp transition with a T(m) of 78.4 degrees C in the presence of a saturating concentration of the divalent cation. The thermal transition observed at intermediate concentrations of divalent metal ions was rather broad and lies between these two extremes of temperature. The equilibrium between the monomer and dimer is dependent on pH, and the optimum for the formation of the dimer shifted from pH 6.0 in the absence of divalent cations to pH 7.5 at saturating concentrations. The beta-lactamase activity increased approximately 2-fold in the presence of saturating concentrations of zinc and cadmium ions. Reaction with beta-lactams caused a shift in the equilibrium toward monomer formation, and thus an apparent inactivation, but the divalent cations protected against this effect.     

Aminoacyl-S-enzyme intermediates in beta-hydroxylations and alpha,beta-desaturations of amino acids in peptide antibiotics

Many of the alpha-amino acids found in proteins are shunted into microbial secondary metabolism to form peptide antibiotics by specific oxidation, including hydroxylation, at the beta carbon. Examples for the enzymatic hydroxylation of tyrosine and histidine and for desaturation of proline during covalent attachment as aminoacyl-S-pantetheinyl enzyme intermediates suggest a general strategy in peptide antibiotic biosynthesis.     

Perspective: transposable elements, parasitic DNA, and genome evolution

The nature of the role played by mobile elements in host genome evolution is reassessed considering numerous recent developments in many areas of biology. It is argued that easy popular appellations such as "selfish DNA" and "junk DNA" may be either inaccurate or misleading and that a more enlightened view of the transposable element-host relationship encompasses a continuum from extreme parasitism to mutualism. Transposable elements are potent, broad spectrum, endogenous mutators that are subject to the influence of chance as well as selection at several levels of biological organization. Of particular interest are transposable element traits that early evolve neutrally at the host level but at a later stage of evolution are co-opted for new host functions.     

Roles of ATP and cytoskeleton in the regulation of Na+/H+ exchanger along the nephron luminal membrane

Although in LLC-PK cells ATP depletion has been shown to result in alterations of cytoskeleton actin and an inhibition of Na+/H+ exchanger activity, there is little information concerning the regulation of this exchanger in the distal luminal membrane by ATP and actin filaments. The present study examined the direct effect of ATP and cytochalasin B on the Na+/H+ exchanger activity in the proximal and distal tubule luminal membranes. The presence of 100 microM ATP in the luminal membrane vesicles from rabbit proximal tubules did not influence the Ethyl Isopropyl Amiloride sensitive Na+ uptake by these membranes. In contrast, the same treatment of luminal membranes from distal tubules significantly enhanced the exchanger activity from 0.22 +/- 0.04 to 0.39 +/- 0.08 pM/microg/10 sec (P < 0.02). When ATP was replaced by its nonhydrolysable form, ATPgammas, the effect on the distal luminal membrane was strongly diminished suggesting that the action of the nucleotide implicates a phosphorylation step. Confirming this hypothesis, addition of 300-microM-Rp cAMP, a protein kinase A inhibitor, completely abolished the effect of ATP. In view of the fact that a tight relationship has been described between ATP, the cytoskeleton complex and the exchanger activity, we studied the effect of cytochalasin B on this activity. The presence of 20 microM cytochalasin B in the distal luminal membrane vesicles induced, as observed with ATP, a significant increase in the Na+ uptake. However, the actions of ATP and cytochalasin B were not additive. These results suggest that firstly, ATP and short actin filaments of the cytoskeleton regulate the distal luminal isoform through an intramembranous mechanism and secondly, a phosphorylation mechanism is, at least partially, implicated in the action of ATP. In contrast, the proximal tubule exchanger is regulated through different mechanisms.     

Autocrine TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in breast cells

In this study, we address whether TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGFbeta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGFbeta. MCF7L TGFbetaRII-transfected cells, which have autocrine TGFbeta activity, were more sensitive to EB1089 than MCF7L cells. TGFbeta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGFbeta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGFbeta isoforms and/or TGFbeta receptors was induced by the analogs in the vitamin D3 and TGFbeta sensitive cells. Vitamin D3 analogs did not induce TGFbeta or TGFbeta receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGFbeta activity through increasing expression of TGFbeta isoforms and/or TGFbeta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGFbeta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGFbeta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGFbeta/vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGFbeta and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGFbeta and vitamin D signaling is also PI 3-kinase pathway dependent.     

Organotin and osmoregulation: quantifying the effects of environmental concentrations of sediment-associated TBT and TPhT on the freshwater-adapted European flounder, Platichthys flesus (L.)

Chronic (5 weeks) exposure of freshwater-adapted European flounder, Platichthys flesus (L.), to environmental concentrations of sediment-associated tri-n-butyltin chloride (TBTCl) and triphenyltin chloride (TPhTCl) caused significant changes to hydromineral fluxes and membrane permeability, mechanisms that maintain osmotic homeostasis. The half-time of exchange of tritiated water (THO) in TBTCl- and TPhTCl-exposed fish was significantly increased during the first 2 weeks of the experiment and then decreased steadily, eventually reaching the level that the control group had constantly maintained throughout the experiment. This change in apparent water permeability was accompanied by a significant decrease in diffusional water flux across the membranes. Passive Na(+)-efflux across the gills was increased significantly but effluxes in the control group were near constant over the same time span. Drinking rates in the organotin groups increased significantly while the rate of urine production did not change. This lead to an increased net water balance in the organotin groups and consequently to a significant reduction of the blood osmolality of both organotin groups when compared to a control. There would appear to be a metabolic cost attached to the changes produced by exposure to environmental levels of organotin compounds which are manifested as a minimal increase in body length compared to the controls.     

Broad programming by IL-2 receptor signaling for extended growth to multiple cytokines and functional maturation of antigen-activated T cells

Coincident production of IL-2 and induction of high-affinity IL-2R upon TCR engagement has precluded a clear distinction for the biological outcome of signaling through TCR/costimulatory molecules vs the IL-2R. Using a novel transgenic mouse on the IL-2Rbeta(-/-) genetic background, this study has separated the relative outcome of signaling through the TCR and IL-2R. We show that stimulation through the TCR and CD28 or CD40 ligand directly leads to T cell activation and several rounds of proliferation in an IL-2-independent fashion. However, this stimulation is insufficient for extended T cell growth to multiple cytokines or differentiation into CTL or IFN-gamma-secreting effector T cells. IL-2 is required for these functions in part by regulation of cyclin D3 and granzyme B. Somewhat less efficiently, IL-4 stimulation of these transgenic T cells redundantly rescued many of these activities. These data demonstrate a fundamental requirement for IL-2 and perhaps other common gamma-chain-dependent cytokines to promote selective gene expression by Ag-activated T cells for their subsequent growth and differentiation into effector T lymphocytes.