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The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3β‐hydroxysteroid dehydrogenase (3β‐HSD) enzymatic activity and gene expression in in vivo and ex vivo systems in pro‐oestrus (PE) and dioestrus I (DI) rats. Animals were injected with vehicle, ALLO, bicuculline or bicuculline plus ALLO and were then killed. In the in vivo experiment, the hypothalamus, ovaries and serum were extracted and analysed. In the ex vivo experiment, the superior mesenteric ganglion ‐ ovarian nerve plexus ‐ ovary system was extracted and incubated during 120 minutes at 37 ºC. The serum and ovarian compartment fluids were used to determine progesterone by radioimmunoanalysis. In the in vivo experiments, ALLO caused a decrease in hypothalamic and ovarian 3β‐HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3β‐HSD activity and gene expression. The ovarian 3β‐HSD activity increased in both stages in the ex vivo system; gene expression increased only during DI. ALLO induced an increase in serum progesterone only in D1 and in the ovarian incubation liquids in both stages. All findings were reversed by an injection of bicuculline before ALLO. Ovarian steroidogenic changes could be attributed to signals coming from ganglion neurones, which are affected by the acute central neurosteroid stimulation. The i.c.v. administration of ALLO via the GABAergic system altered 3β‐HSD activity and gene expression, modulating the neuroendocrine axis. The present study reveals the action that ALLO exerts on the GABAA receptor in both the central and peripheral nervous system and its relationship with hormonal variations. ALLO is involved in the "fine tuning" of neurosecretory functions as a potent modulator of reproductive processes in female rats. [ABSTRACT FROM AUTHOR]

Abstract: Background: Despite suffering high ultraviolet radiation levels, few data on malignant melanoma (MM) in Macaronesia are available. Methods: Observational study of cutaneous MM cases diagnosed during a period of 12 years at a tertiary hospital in Canary Islands. Results: A total of 532 patients (female/male = 1.4) with an average age of 56 years were included; 5% developed more than one MM, and 7% reported family history of MM. Phototype II (43%), dark eyes (41%), and dark hair (41%) predominated. There was a lower frequency of light‐colored hair and eyes in those born in the Canary Islands. The most frequent locations of MM were on the back for men (37%) and on the lower extremities for women (35%). Among the infiltrating tumors (83%), the (median) thickness was 1.07 mm (women, 0.90 mm; men, 1.21 mm). Anatomopathological ulceration (AU) and a mitotic rate ≥1 mitosis/mm2 (HMR) were recorded in 27% of patients. Patients with regional disease constituted 12% of the population. The most common stage was IA (34%). Melanoma‐specific survival (MSSV) decreased significantly with thickness, presence of AU, HMR, and sentinel lymph node disease. These four variables were independent prognostic factors. The five‐year MSSV varied between 100% (stage IA) and 39% (stage IIIC). Conclusions: The characteristics of the patients were similar to those published in datasets from continental Europe, although the pigmentary features were darker in those originating from Macaronesia. The prognostic parameters described in the 7th edition of the American Joint Committee on Cancer (AJCC) independently predict MSSV in our patients. [ABSTRACT FROM AUTHOR]

It has been shown that a single exposure to amphetamine is sufficient to induce long-term behavioral, neurochemical, and neuroendocrine sensitization in rats. Dopaminergic neurotransmission in the nucleus accumbens and the caudateputamen plays a critical role in the addictive properties of drugs of abuse. Angiotensin (Ang) II receptors are found on the soma and terminals of mesolimbic dopaminergic neurons and it has been shown that Ang II acting through its AT1 receptors facilitates dopamine release. The hypothesis was tested that Ang II AT1 receptors are involved in the neuroadaptative changes induced by a single exposure to amphetamine and that such changes are related to the development of behavioral and neurochemical sensitization. For this purpose, the study examined the expression of amphetamine-enhanced (0.5 mg kg-1 i.p.) locomotor activity in animals pretreated with candesartan, an AT1 blocker, (3 mg kg-1 p.o 3 5 days), 3 weeks after an amphetamine injection (5 mg kg-1 i.p.). Dopaminergic hyperreactivity was tested by measuring the 3H-DA release in vitro from caudate-putamen and nucleus accumbens slices, induced by K+ stimulus. It was confirmed the behavioral sensitization in the two-injection protocoland candesartan pretreatment attenuate this response. It was also found that AT1 blockade pretreatment did not affect the locomotor response to dopamine agonists. In respect to the neurochemical sensitization tested using ex vivo 3H-DA release experiments it was found that AT1 receptor pretreatment blunted the enhanced response induced by K+ stimulus. The results support the idea that the development of neuroadaptive changes induced by amphetamine involves brain AT1 Ang II receptor activation. [ABSTRACT FROM AUTHOR]

Abstract: In a previous work, chemical modification of Cys-238 of Escherichia coli Pfk-2 raised concerns on the importance of the dimeric state of Pfk-2 for enzyme activity, whereas modification of Cys-295 impaired the enzymatic activity and the MgATP-induced tetramerization of the enzyme. The results presented here demonstrate that the dimeric state of Pfk-2 is critical for the stability and the activity of the enzyme. The replacement of Cys-238 by either Ala or Phe shows no effect on the kinetic parameters, allosteric inhibition, dimer stability and oligomeric structure of Pfk-2. However, the mutation of Cys-295 by either Ala or Phe provokes a decrease in the k cat value and an increment in the K m values for both substrates. We suggest that the Cys-295 residue participates in intersubunit interactions in the tetramer since the Cys-295-Phe mutant exhibits higher tetramer stability, which in turn results in an increase in the fructose-6-P concentration required for the reversal of the MgATP inhibition relative to the wild type enzyme. [Copyright &y& Elsevier]

Escherichia coli contains two phosphofructokinases, Pfk-1 and Pfk-2, which belong to unrelated protein families. In addition to catalytic function, the enzymes have converged in showing substrate inhibition by the nucleotide MgATP. However, although both Pfk-1 and Pfk-2 have been extensively characterized biochemically, only the structure of the former has been solved by X-ray diffraction. In order to fully understand how the same function has evolved on different structural folds, Pfk-2 has been crystallized by the hanging-drop vapour-diffusion method using PEG 6000 as precipitant. Single crystals were grown in the presence of MgATP and diffracted to 1.98 Å. The crystals belong to the orthorhombic system, space group P2221, with unit-cell parameters a = 42.8, b = 86.8, c = 171.3 Å. The calculated Matthews coefficient of 2.45 Å3 Da−1 indicates the presence of two monomers in the asymmetric unit, corresponding to a solvent content of 49%. Structure determination is ongoing. [ABSTRACT FROM AUTHOR]