Native ion current coupled to purinergic activation via basal and mechanically induced ATP release in Xenopus follicles

Xenopus follicle-enclosed oocytes are endowed with purinergic receptors located in the follicular cell membrane; their stimulation by ATP elicits an electrical response that includes generation of a fast inward current (F(Cl)) carried by Cl(-). Here, it was found that mechanical stimulation of the follicle provoked a native electrical response named I(mec). This was dependent on coupling between oocyte and follicular cells, because I(mec) was eliminated by enzymatic defolliculation or application of uncoupling drugs, such as heptanol or carbenoxolone. Moreover, the characteristics of I(mec) suggested that it corresponded with opening of the Cl(-) channel involved in F(Cl). For example, I(mec) showed cross-talk with the membrane mechanism that activates the F(Cl) response and anionic selectivity similar to that displayed by F(Cl). Also like F(Cl), I(mec) was independent of extracellular or intracellular Ca(2+). Furthermore, I(mec) was inhibited by superfusion with a purinergic antagonist, suramin, or by an enzyme that rapidly hydrolyzes ATP, apyrase. The response to mechanical stimulation was reconstituted in defolliculated oocytes expressing P2X channels as an ATP sensor. Recently, it has been shown that ATP release from the Xenopus oocyte is triggered by mechanical stimulation. Together, these observations seemed to indicate that I(mec) is activated through a mechanism that involves oocyte release of ATP that diffuses and activates purinergic receptors in follicular cells, with subsequent opening of F(Cl) channels. Thus, I(mec) generation disclosed a paracrine communication system via ATP between the oocyte and its companion follicular cells that might be of physiological importance during the growth and development of the gamete.     

An apoptosis-inducing serine protease secreted by the entomopathogenic nematode Steinernema carpocapsae

Steinernema carpocapsae is an insect parasitic nematode able to parasitise and kill the host within 48 h. Secreted products (ESP) of the parasitic stage of a virulent strain contain higher amounts of proteolytic activity than a low virulence strain, suggesting proteases are involved in virulence. From the ESP we purified a protein (Sc-SP-3) with a M_r of 30 kDa and a pI of 7 that cleaved the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA and was inhibited by phenylmethanesulfonyl fluoride, benzamidine and chymostatin, thus indicating that it belongs to the chymotrypsin-like serine protease family. Sc-SP-3 has a V_max of 0.3 mM min⁻¹ ml⁻¹ and K_m of 6.6 × 10⁻⁴ M, with maximum activity at pH 8 and 40 °C. The full-length cDNA was obtained using degenerate oligonucleotides for serine proteases. This open reading frame encodes a preproprotein containing a putative signal peptide composed of 16 amino acid residues, a prodomain of 40 residues and a mature protease domain of 261 residues, including the catalytic triad His/Asp/Ser characteristic of trypsin-like serine proteases. The N-terminal sequence and the peptide masses fingerprint obtained by MALDI-TOF–MS for the purified protein matched the cDNA. Gene expression analysis by quantitative real-time-PCR showed that this gene is expressed only during the parasitic stage and that pre-invasive nematodes inside the mid-gut expressed higher amounts of Sc-SP-3 than those that already enter the haemocoel. Sc-SP-3 caused histolysis in the insect mid-gut. In vitro assays demonstrated that Sc-SP-3 digested extracellular proteins and induced apoptosis in Sf9 insect cells, thus suggesting Sc-SP-3 is a multifunctional chymotrypsin-like protease involved in pathogenesis.     

Diverse endophytic bacteria isolated from a leguminous tree Conzattia multiflora grown in Mexico

Conzattia multiflora is a leguminous tree present only in Mexico and Guatemala. There is no record about its symbiotic or pathogenic microbes. In this study, we found that numerous bacteria with 10⁴–10⁶ individuals per gram of fresh epidermis were distributed in the tissue of this plant. All the bacteria isolated from the Conzattia epidermis were Gram-negative, facultative anaerobic rods and formed yellow or colorless colonies. They were identified as endophytes by inoculation tests. Some of the bacteria could significantly promote the growth of Conzattia seedlings. Nine different groups were defined by PCR-based RFLP, which were classified as Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella by the phylogenetic analysis of 16S rRNA genes. The existence of plant-borne lineages of Salmonella indicates that the unexplored plants may harbor some unknown microbes.     

Age-dependent axonal expression of potassium channel proteins during development in mouse hippocampus

The development of the hippocampal network requires neuronal activity, which is shaped by the differential expression and sorting of a variety of potassium channels. Parallel to their maturation, hippocampal neurons undergo a distinct development of their ion channel profile. The age-dependent dimension of ion channel occurrence is of utmost importance as it is interdependently linked to network formation. However, data regarding the exact temporal expression of potassium channels during postnatal hippocampal development are scarce. We therefore studied the expression of several voltage-gated potassium channel proteins during hippocampal development in vivo and in primary cultures, focusing on channels that were sorted to the axonal compartment. The Kv1.1, Kv1.2, Kv1.4, and Kv3.4 proteins showed a considerable temporal variation of axonal localization among neuronal subpopulations. It is possible, therefore, that hippocampal neurons possess cell type-specific mechanisms for channel compartmentalization. Thus, age-dependent axonal sorting of the potassium channel proteins offers a new approach to functionally distinguish classes of hippocampal neurons and may extend our understanding of hippocampal circuitry and memory processing.     

Study of benzo[a]phenazine 7,12-dioxide as selective hypoxic cytotoxin-scaffold. Identification of aerobic-antitumoral activity through DNA fragmentation

Phenazine 5,10-dioxides are prodrugs for antitumor therapy that undergo hypoxic-selective bioreduction to form cytotoxic species. Here we investigate the expanded system benzo[a]phenazine 7,12-dioxides as selective hypoxic cytotoxin-scaffold. The clonogenic survival of V79 cells on aerobic and anaerobic conditions, conduct us to study antiproliferative activity on Caco-2 tumoral cells in normoxia. Electrochemical, DNA-interaction and DNA-damage studies were performed to establish the mode of action. The results demonstrated the potential biological properties of the studied scaffold being derivatives 6–10 structural hits for further chemical-modifications to become into therapeutics for solid tumors. Compounds 6 and 8 with cytotoxicity against V79 cells in both conditions (aerobia and anaerobia) were also cytotoxic against Caco-2 tumoral cells in aerobiosis.     

Mitochondrial ryanodine receptors and other mitochondrial Ca permeable channels

Ca²⁺ channels that underlie mitochondrial Ca²⁺ transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca²⁺ uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca²⁺ transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca²⁺ overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca²⁺ channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca²⁺ channels.     

Zinc and Fatty Acids in Depression

Polyunsaturated Fatty acids (PUFAs) seem to be helpful in the therapy of depression. Zinc (Zn) may be one co-factor contributing to their antidepressive effect. Zn acts lipid protective and is a constituent of fatty acid metabolism enzymes. In animals, an antidepressive effect of Zn was already demonstrated. Therefore, if and how Zn and PUFAs correlate in depressive patients or in individuals from the general population was investigated. Blood samples were collected from 88 depressive in-patients and 88 volunteers from the general population matched for age-group and gender (each 32 men and 56 women, 21–70 years) for measurement of Zn (colorimetric) and of 12 fatty acids (FAs) (by capillary gas-chromatography). Severity of depression in patients was assessed by Beck Depression Inventory (BDI) and Hamilton Depression Rating Scale (HDRS). Zn concentration was independent of age, gender and body-mass-index and significantly correlated with the severity of depression measured by BDI (r = 0.26; P = 0.034) in depressive patients,. HDRS was inversely correlated with gammalinolenic acid concentration (r = −0.24; P = 0.029). Median serum Zn concentration in depressive patients did not differ from control individuals. Zn was correlated with myristic acid concentration (r = 0.22; P < 0.05) in controls from the general population; and a negative correlation between Zn and dihomogammalinolenic acid concentration (r = −0.26; P < 0.05) was found in depressive inpatients. FA composition in serum significantly differed between depressive and healthy persons: Depressive patients had higher stearic and arachidonic acid (AA) concentration. Relative to AA, their eicosapentaenoic and docosapentaenoic acid concentration were diminished compared to the general populations group. These results do not confirm the hypothesis of a general lack of Zn in depressive disorders, but Zn concentrations differed dependent on comorbid disorders and severity of depression. In depressive patients and control persons Zn concentration is associated with different FAs indicating diverging metabolic pathways.     

Free Radicals and Carcinogenesis

The role of free radicals and active states of oxygen in human cancer is as yet unresolved. Various lines of evidence provide strong but inferential evidence that free radical reactions can be of crucial importance in certain carcinogenic mechanisms. A central point in considering free radical reactions in carcinogenesis is that human cancer is really a group of highly diverse diseases for which the initial causation and the progression to clinical disease occur through a wide variety of mechanisms. Furthermore, for many human cancers it appears that there are alternate pathways capable of tumor initiation and tumor progression. While for certain of these pathways free radical reactions appear necessary, it is unlikely that there are human cancers for which free radicals, or any other mechanism, are sufficient for the entire processbeginning with the genetic alteration leading to a somatic mutation and eventually resulting in clinically overt disease. It is crucial that we view free radical reactions as aong a panoply of mechanisms leading to human cancer, and consider research about the role of free radicals in cancer as opportunities to prevent the initiation or progression of human cancer.