Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus

The intracellular localization and activity of the nitric oxide synthase (NOS) isoforms were investigated in rat brown adipocytes. Immunohistochemistry showed cytoplasmic and nuclear staining for the endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms; accordingly, anti-L-citrulline antibody, a marker of NOS activity, immunostained both the cytoplasm and the nucleus. The presence of metabolically active NOS in the nucleus was further confirmed by immunoblotting analyses of subcellular fractions of homogenates from cultured brown adipocytes and by measurements of NOS activity in the cytosol and nucleus. Sympathetic stimulation in vivo (i.e. cold exposure or beta(3)-adrenergic agonist treatment) and in vitro (i.e. noradrenaline treatment of cultured cells) significantly increased both cytosolic and nuclear eNOS and iNOS expression and activities. By contrast, the number of iNOS-positive, but not eNOS-positive, nuclei was significantly lower in the functionally impaired brown fat of genetically obese Zucker fa/fa rats. These data suggest the existence of a noradrenaline-modulated functional NOS system in the nucleus of brown adipocytes.     

Effect of dimethyl sulfoxide on the structure and the functional properties of horseradish peroxidase as observed by spectroscopy and cyclic voltammetry

Electrochemical biosensors have found wide application in food and clinical areas, as well as in environmental field. A large number of articles focused on horseradish peroxidase (HRP)-based biosensors have been published in the last decade, due to the capability of HRP to quantitatively detect the presence of hydrogen peroxide produced in a reaction. At present a large body of multi-enzymatic amperometric biosensors are realized by entrapping HRP together with other enzymes into a polymeric matrix; these systems represent a promising example of simple, low-cost electrochemical tools for the analysis of bioanalytes in solution, such as glucose, choline and cholesterol. Due to the fact that polymers used for HRP entrapping are soluble in organic solvents and that many solvents are strong denaturants of aquo-soluble proteins, in this paper we investigate (in particular, by circular dichroism and electron paramagnetic spectroscopies) the effect of dimethyl sulfoxide, one of the organic solvents employed for polymer solubilization, on the structure and the functionality of HRP, in order to determine the effect induced by the solvent concentration on the structure and activity of the hemoprotein. This is relevant for basic and applied biochemistry, HRP being largely employed in bioinorganic chemistry and sensor area.     

Regulation of niemann-pick c1 gene expression by the 3'5'-cyclic adenosine monophosphate pathway in steroidogenic cells

The Niemann Pick-C1 (NPC-1) protein is essential for intracellular transport of cholesterol derived from low-density lipoprotein import in mammalian cells. The role of the protein kinase A (PKA) pathway in regulation of expression of the NPC-1 gene was investigated. NPC-1 promoter activity was induced by treatment with dibutryl cAMP (dbcAMP), alone or in combination with the cAMP response element (CRE) binding protein (CREB) overexpressed in adrenal Y-1 cells. When the catalytic subunit of PKA was overexpressed in Y-1 cells, there were similar increases in NPC-1 promoter activity in the presence of CREB. Responses were attenuated by blockade of the PKA pathway, and in the Kin-8 cell line deficient in PKA. Promoter deletion analysis revealed that this response was present in promoter fragments of 186 bp and larger but not present in the 121-bp fragment. Two promoter regions, one at -430 and one at -120 upstream of the translation initiation site, contained CRE consensus sequences. These bound recombinant CREB in EMSA, confirming their authenticity as CREB response elements. Promoters bearing mutations of both CRE displayed no response to dbcAMP. The orphan nuclear receptor, steroidogenic factor-1 (SF-1), was implicated in NPC-1 transactivation by the presence of SF-1 target sequence that formed a complex with recombinant SF-1 in EMSA. Furthermore, transfection of a plasmid that overexpressed SF-1 into ovarian granulosa cells increased promoter activity in response to dbcAMP, an effect abrogated by mutation of the SF-1 target sequence. Chromatin immunoprecipitation assays demonstrated that the CRE region of the endogenous and transfected NPC-1 promoter associated with both acetylated and phosphorylated histone H-3 and that this association was increased by dbcAMP treatment. Treatment with dbcAMP also increased the association of the CRE region of the promoter with CREB binding protein, which has histone acetyltransferase activity. Together, these results demonstrate a mechanism of regulation of NPC-1 expression by the cAMP-PKA pathway that includes PKA phosphorylation of CREB, recruitment of the coactivator CREB binding protein and the phosphorylation and acetylation of histone H-3 to transactivate the NPC-1 promoter.     

Synthesis of (20S)-[7,7,21,21-2H4]-3beta-(tert-butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol,a useful intermediate for the preparation of deuterated isotopomers of sterols

(20S)-[7,7,21,21-2H(4)]-3beta-(tert-Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, an intermediate for the preparation of deuterated isotopomers of sterols to be used as standards for biomedical studies, was prepared by reduction with dichloroaluminum deuteride of ethyl (20S)-3beta-(tert-butyldimethylsilanyloxy)-7-oxo-pregn-5-en-20-carboxylate. Using controlled experimental conditions, it has also been shown that the dichloroaluminum hydride reduction of a 7-keto steroid affords the corresponding 7beta-hydroxy derivative in a highly stereoselective manner.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with lipophilic sulfonamides

A series of new compounds was obtained by reaction of aromatic/heterocyclic sulfonamides incorporating amino groups with N,N-diphenylcarbamoyl chloride and diphenylacetyl chloride. These sulfonamides were assayed for the inhibition of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic CA I and CA II, and the transmembrane, cancer-associated isozyme CA IX. Good inhibitors against all these isoforms were detected, and the inhibition profile of the newly investigated isozyme IX was observed to be different from that of the cytosolic isozymes, I and II. This may lead to the development of novel anticancer therapies based on the selective inhibition of CA IX.     

Effect of bradykinin analogues on the B1 receptor of rat ileum

The longitudinal muscle of isolated rat ileum is a sensitive bioassay suitable for testing compounds with antagonistic effects on the B(1) receptor. Bradykinin analogues with replacement of proline by alkyl-substituted phenylalanine at position 7 are effective on this receptor as entire molecules and have a stronger antagonistic effect than on the B(2) receptor. A corresponding desArg(9)-compound has a specific effect on the B(1) receptor and a very high antagonistic potency. [LNMPhe(2)]bradykinin as a compound without any replacement at position 7 or 8 shows antagonistic activity as well.     

Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri

The cellular compartmentation of elements was analysed in the Zn hyperaccumulator Arabidopsis halleri (L.) O'Kane & Al-Shehbaz (=Cardaminopsis halleri) using energy-dispersive X-ray microanalysis of frozen-hydrated tissues. Quantitative data were obtained using oxygen as an internal standard in the analyses of vacuoles, whereas a peak/background ratio method was used for quantification of elements in pollen and dehydrated trichomes. Arabidopsis halleri was found to hyperaccumulate not only Zn but also Cd in the shoot biomass. While large concentrations of Zn and Cd were found in the leaves and roots, flowers contained very little. In roots grown hydroponically, Zn and Cd accumulated in the cell wall of the rhizodermis (root epidermis), mainly due to precipitation of Zn/Cd phosphates. In leaves, the trichomes had by far the largest concentrations of Zn and Cd. Inside the trichomes there was a striking sub-cellular compartmentation, with almost all the Zn and Cd being accumulated in a narrow ring in the trichome base. This distribution pattern was very different from that for Ca and P. The epidermal cells other than trichomes were very small and contained lower concentrations of Zn and Cd than mesophyll cells. In particular, the concentrations of Cd and Zn in the mesophyll cells increased markedly in response to increasing Zn and Cd concentrations in the nutrient solution. This indicates that the mesophyll cells in the leaves of A. halleri are the major storage site for Zn and Cd, and play an important role in their hyperaccumulation. Received: 4 April 2000 / Accepted: 16 May 2000     

Tyrosine Phosphorylation and Src Family Kinases Control Keratinocyte Cell–Cell Adhesion

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that β-catenin, γ-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of α-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of α-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of β- and γ-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn−/− mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of β-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.     

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.     

Non-invasive Parenchymal,Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.