Norepinephrine transporter knockout-induced up-regulation of brain alpha2A/C-adrenergic receptors

The norepinephrine transporter (NET) is responsible for the rapid removal of norepinephrine released from sympathetic neurons; this release is controlled by inhibitory alpha(2)-adrenergic receptors (alpha(2)ARs). Long-term inhibition of the NET by antidepressants has been reported to change the density and function of pre- and postsynaptic ARs, which may contribute to the antidepressant effects of NET inhibitors such as desipramine. NET-deficient (NET-KO) mice have been described to behave like antidepressant-treated mice. By means of quantitative real-time PCR we show that mRNAs encoding the alpha(2A)-adrenergic receptor (alpha(2A)AR) and the alpha(2C)-adrenergic receptor (alpha(2C)AR) are up-regulated in the brainstem, and that alpha(2C)AR mRNA is also elevated in the hippocampus and striatum of NET-KO mice. These results were confirmed at the protein level by quantitative autoradiography. The NET-KO mice showed enhanced binding of the selective alpha(2)AR antagonist [(3)H]RX821002 in several brain regions. Most robust increases (20-25%) in alpha(2)AR expression were observed in the hippocampus and in the striatum. Significant increases (16%) were also seen in the extended amygdala and thalamic structures. In an 'in vivo' test, the alpha(2)AR agonist clonidine (0.1 mg/kg) caused a significantly greater reduction of locomotor activity in NET-KO mice than in wild-type mice, showing the relevance of our findings at the functional level.     

In vitro versus in vivo cellulose microfibrils from plant primary wall synthases: structural differences

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry) were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (1-->3)-beta-d-glucan (callose). The taurocholate extracts, which did not require the addition of Mg(2+), were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IV(I), a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.     

Mac-1 (CD11b/CD18) as accessory molecule for Fc alpha R (CD89) binding of IgA

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.     

Incision of damaged versus nondamaged DNA by the Escherichia coli UvrABC proteins.

Incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires the UvrA, UvrB, and UvrC proteins as well as ATP hydrolysis. This incision reaction can be divided into three steps: site recognition, preincision complex formation, and incision. UvrAB is able to execute the first two steps in the reaction while the addition of UvrC is required for the incision of DNA. This incision reaction does not require ATP hydrolysis and results in the formation of a tight UvrABC post-incision complex and the generation of an oligomer of approximately 12 nucleotides. At high UvrABC concentrations the specificity of the incision for damaged DNA is decreased and significant incision of undamaged DNA occurs. Analogous to damage specific incision, this type of incision leads to generation of an oligonucleotide, but in this case the size is approximately 9 nucleotides in length. Further evidence shows that the combination of UvrB and UvrC proteins can generate a significant amount of a similar size product on undamaged DNA. In addition, the UvrC protein alone can generate a small amount of the same product. Immunological characterization of the weak nuclease activity seen with UvrC indicates that the activity is very tightly associated with the purified UvrC protein.     

Subunit organization of PSI particles from brown algae and diatoms: polypeptide and pigment analysis

P700 enriched fractions were isolated from two brown algae and one diatom using sucrose density centrifugation after digitinin solubilization. They had a Chl a/P700 ratio of about 250 to 375 according to the species, they were enriched in long-wavelength absorbing Chl a and exhibited a fluorescence emission maximum at 77 K near 720 nm. They all presented a major polypeptide component at 66±2 kDa, but their polypeptide composition was rather complex and somewhat different from one species to another. Further solubilization with dodecylmaltoside of those native PSI particles allowed the separation of two or three fractions. The lightest, xanthophyll-rich, fraction was identified to be a light-harvesting complex. It contained no P700 and had a major polypeptide of molecular weight near 20 kDa (at the same molecular weight than the respective LH native fraction of each species) and exhibited a 77 K peak fluorescence emission at 685 nm. The other fractions were enriched in P700 and almost entirely depleted in xanthophylls. When two of them are present, they both exhibited a major polypeptide at 66±2 kDa and were totally devoid of the LH polypeptide, but the two fractions widely differed one from another in the abundance and molecular weight of the other polypeptide components. The most purified of these two fractions presented a composition similar to PSI core complex from green plants.Abbreviations LH light-harvesting - LHCII light-harvesting complex II of green plants - P700 reaction center chlorophyll of PSI     

Crystal structure of the copper-containing quercetin 2,3-dioxygenase from Aspergillus japonicus

Quercetin 2,3-dioxygenase is a copper-containing enzyme that catalyzes the insertion of molecular oxygen into polyphenolic flavonols. Dioxygenation catalyzed by iron-containing enzymes has been studied extensively, but dioxygenases employing other metal cofactors are poorly understood. We determined the crystal structure of quercetin 2,3-dioxygenase at 1.6 A resolution. The enzyme forms homodimers, which are stabilized by an N-linked heptasaccharide at the dimer interface. The mononuclear type 2 copper center displays two distinct geometries: a distorted tetrahedral coordination, formed by His66, His68, His112, and a water molecule, and a distorted trigonal bipyramidal environment, which additionally comprises Glu73. Manual docking of the substrate quercetin into the active site showed that the different geometries of the copper site might be of catalytic importance.     

Facteurs impliqués dans le remodelage de la chromatine au cours de la spermiogenèse

Round spermatids are post-meiotic cells with a haploid genome contained in a nucleus, with a structure initially similar to that of the somatic cell nucleus. During spermatogenesis, the spermatid nucleus undergoes drastic remodelling during which it first elongates and then condenses into the very specific and tightly packaged structure of the sperm nucleus. During this remodelling dthe histones are replaced by transition proteins, which, in turn, are replaced by protamines, the specific nuclear proteins of the spermatozoa. Immediately prior to their replacement, the histones are hyperacetylated. The first part of our work was to precisely characterise the changes in histone acetylation during murine spermatogenesis. We have shown that the core histones H2A, H2B, H3 and H4 are hyperacetylated in the elongating spermatids. We have also shown that these changes in acetylation are associated with degradation of the enzymes responsible for histone deacetylation, histone deacetylases or HDACs, while histone acetyl transferases are still present in these cells. The histone acetylation pattern was also investigated during human spermatogenesis, revealing that histone hyperacetylation in the nucleus of elongating spermatids, which appears to be conserved during the course of evolution, also occurs during human spermatogenesis. Moreover, our data obtained from the testes of men with severely altered spermatogenesis, including SCO syndromes (Sertoli Cells Only Syndromes), show that a global hyperacetylation of the Sertoli cell nuclei is associated with an absence of meiotic and post-meiotic cells. This suggests that the global histone acetylation variations observed during spermatogenesis are part of a signalling pathway involving germ cell — Sertoli cell communication. Altogether, these data provide a basis for a better understanding of the mechanisms and identification of the factors involved in post-meiotic remodelling of chromatin.     

Towards the establishment of a porcine model to study human amebiasis

Background

Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis.

Methodology/Principal Findings

We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma.

Conclusions

The pig model could help with simultaneously studying intestinal and extraintestinal lesion development.     

The mammalian beta 2-adrenergic receptor: purification and characterization

The beta 2-adrenergic receptors from hamster, guinea pig, and rat lungs have been solubilized with digitonin and purified by sequential Sepharose-alprenolol affinity and high-performance steric-exclusion liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveal a peptide with an apparent Mr of 64 000 in all three systems that coincides with the peptide labeled by the specific beta-adrenergic photoaffinity probe (p-azido-m-[125I]iodobenzyl)carazolol. A single polypeptide was observed in all three systems, suggesting that lower molecular weight peptides identified previously by affinity labeling or purification in mammalian systems may represent proteolyzed forms of the receptor. Purification of the beta-adrenergic receptor has also been assessed by silver staining, iodinated lectin binding, and measurement of the specific activity (approximately 15 000 pmol of [3H]dihydroalprenolol bound/mg of protein). Overall yields approximate 10% of the initial crude particulate binding, with 1-3 pmol of purified receptor obtained/g of tissue. The purified receptor preparations bind agonist and antagonist ligands with the expected beta 2-adrenergic specificity and stereoselectivity. Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.     

Relationship between Longitudinal Continuity of Primary Care and Likelihood of Death: Analysis of National Insurance Data

Background

Continuity of care (COC) is a widely accepted core principle of primary care and has been associated with patient satisfaction, healthcare utilization and mortality in many, albeit small, studies.

Objective

To assess the relationship between longitudinal continuity with a primary care physician (PCP) and likelihood of death in the French general population.

Design

Observational study based on reimbursement claims from the French national health insurance (NHI) database for salaried workers (2007–2010).

Setting

Primary care.

Patients

We extracted data on the number and pattern of visits made to a PCP and excluded all patients who did not visit a PCP at least twice within 6 months. We recorded age, gender, comorbidities, social status, and deaths.

Main outcome measures

The primary endpoint was death by all causes. We measured longitudinal continuity of care (COC) with a PCP twice a year between 2007 and 2010, using the COC index developed by Bice and Boxerman. We introduced the COC index as time-dependent variables in a survival analysis after adjustment for age, gender and stratifying on comorbidities and social status.

Results

A total of 325 742 patients were included in the analysis. The average COC index ranged from 0.74 (SD: 0.35) to 0.76 (0.35) (where 1.0 is perfect continuity). Likelihood of death was lower in patients with higher continuity (hazard ratio for an increase in 0.1 of continuity, adjusted for age, sex, and stratified on comorbidities and social status: 0.96 [0.95–0.96]).

Conclusion

Higher longitudinal continuity was associated with a reduced likelihood of death.