Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.

Beta-endorphin cell therapy for cancer prevention.

ß-Endorphin (BEP)-producing neuron in the hypothalamus plays a key role in bringing the stress axis to a state of homeostasis and maintaining body immune defense system. Long-term delivery of BEP to obtain beneficial effect on chemoprevention is challenging, as the peptides rapidly develop tolerance. Using rats as animal models, we show here that transplantation of BEP neurons into the hypothalamus suppressed carcinogens- and hormone-induced cancers in various tissues and prevented growth and metastasis of established tumors via activation of innate immune functions. In addition, we show that intracerebroventricular administration of nanosphere-attached dibutyryl cyclic adenosine monophosphate (dbcAMP) increased the number of BEP neurons in the hypothalamus, reduced the stress response, enhanced the innate immune function, and prevented tumor cell growth, progression, and metastasis. BEP neuronal supplementation did not produce any deleterious effects on general health but was beneficial in suppressing age-induced alterations in physical activity, metabolic, and immune functions. We conclude that the neuroimmune system has significant control over cancer growth and progression, and that activation of the neuroimmune system via BEP neuronal supplementation/induction may have therapeutic value for cancer prevention and improvement of general health.

Inhibition of phosphodiesterase-4 promotes oligodendrocyte precursor cell differentiation and enhances CNS remyelination.

The increasing effectiveness of new disease-modifying drugs that suppress disease activity in multiple sclerosis has opened up opportunities for regenerative medicines that enhance remyelination and potentially slow disease progression. Although several new targets for therapeutic enhancement of remyelination have emerged, few lend themselves readily to conventional drug development. Here, we used transcription profiling to identify mitogen-activated protein kinase (Mapk) signalling as an important regulator involved in the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes. We show in tissue culture that activation of Mapk signalling by elevation of intracellular levels of cyclic adenosine monophosphate (cAMP) using administration of either dibutyryl-cAMP or inhibitors of the cAMP-hydrolysing enzyme phosphodiesterase-4 (Pde4) enhances OPC differentiation. Finally, we demonstrate that systemic delivery of a Pde4 inhibitor leads to enhanced differentiation of OPCs within focal areas of toxin-induced demyelination and a consequent acceleration of remyelination. These data reveal a novel approach to therapeutic enhancement of remyelination amenable to pharmacological intervention and hence with significant potential for translation.

Interactions of cyclic AMP and its dibutyryl analogue with model membrane: X-ray diffraction and Raman spectroscopic study using cubic liquid-crystalline phases of monoolein.

Interactions of adenosine 3':5'-cyclic monophosphate (cAMP) and its dibutyryl analogue, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP), with a lipid bilayer were studied by small-angle X-ray diffraction (SAXD) and Raman spectroscopy. The cubic Pn3m phase of monoolein (MO) served as a bilayer-based model system. SAXD measurements have indicated that incorporation of approximately 3 wt.% cAMP leaves the phase parameters practically unaltered, whereas the same content of dbcAMP induces the intercubic Pn3m-->Ia3d transition. By applying the concepts of lipid shape parameter and infinite periodic minimal surface to these MO phases, we have suggested that, as opposed to cAMP, dbcAMP associates with the MO bilayer. This conclusion has been supported by the different effects of phase matrix on the Raman shifts of the adenine and phosphate vibrational modes of these two nucleotides. Moreover, Raman spectra have indicated that dbcAMP inserts into the bilayer through the butyryladenine group, positioning dbcAMP preferentially at the polar/apolar interface.

Precursors of the purine backbone augment the inhibitory action of hypoxanthine and dibutyryl cAMP on mouse oocyte maturation.

In this study we have tested the hypothesis that precursors of the purine base backbone--glutamine, glycine, aspartic acid, and formate--promote meiotic arrest when included in medium containing established meiotic inhibitors and that this occurs in glucose-dependent fashion. An initial experiment established that in medium supplemented with 4 mM hypoxanthine and containing no purine precursors, very little meiotic arrest was maintained in cumulus cell-enclosed oocytes after 17-18 hr (90% germinal vesicle breakdown; GVB). Increasing concentrations of glucose reduced the maturation percentage such that only 57% had matured at 0.55 mM. The addition of 2 mM glutamine (Gln) alone reduced the maturation percentage in the absence of glucose (70% GVB), and the further addition of glucose revealed an additive inhibitory effect between these two supplements. Dose response experiments with Gln, glycine (Gly), aspartic acid and formate showed that in medium supplemented with hypoxanthine, very little inhibitory action was observed in the absence of glucose but that upon addition of this hexose, a dramatic decrease in maturation percentage was observed in the Gln and Gly groups. Results of experiments using combinations of precursors showed that when Gln and Gly were added together, greater augmentation of meiotic arrest maintained by either hypoxanthine or dibutyryl cAMP was achieved in the presence of glucose than with either amino acid alone. The addition of purine precursors significantly increased the extent of purine nucleotide production by oocyte-cumulus cell complexes, and this was accentuated by glucose. It is concluded that the presence of purine precursors can augment the meiosis-arresting action of established meiotic inhibitors in glucose-dependent fashion, and that this is due, at least in part, to their incorporation into purine nucleotides via the de novo synthetic pathway.

Regulation of calcium-activated nonselective cation channel activity by cyclic nucleotides in the rat insulinoma cell line, CRI-G1.

The regulation of a calcium-activated nonselective cation (Ca-NS+) channel by analogues of cyclic AMP has been investigated in the rat insulinoma cell line, CRI-G1. The activity of the channel is modulated by cyclic AMP in a complex way. In the majority of patches (83%) tested concentrations of cyclic AMP of 10 microM and above cause an inhibition of channel activity which is immediately reversible on washing. In contrast, lower concentrations of cyclic AMP, between 0.1 and 1.0 microM, produce a transient activation of channel activity in most patches (63%) tested. One group of analogues, including N6-monobutyryl cyclic AMP and N6, 2'-O-dibutyryl cyclic AMP reduced the activity of the Ca-NS+ channel at all concentrations tested and 2'-O-Monobutyryl cyclic AMP produced inhibition in all patches tested except one, at all concentrations. A second group produced dual concentration-dependent effects on Ca-NS+, low concentrations stimulating and high concentrations inhibiting channel activity. 6-Chloropurine cyclic AMP and 8-bromo cyclic AMP produced effects similar to those of cyclic AMP itself. In contrast, 8-[4-chlorophenylthio] cyclic AMP also showed a dual action, but with a high level of activation at all concentrations tested up to 1 mM. Ca-NS+ channel activity was also predominantly activated by low concentrations of Sp-cAMPS. The activating effects of both Sp-cAMPS and cyclic AMP are antagonized by Rp-cAMPS, which by itself only produced a weak inhibition of Ca-NS+ channel activity even at concentrations of 10 microM and above. The results are discussed in terms of a model in which cyclic AMP, and other cyclic nucleotides, modulate the activity of the Ca-NS+ channel by binding to two separate sites.