Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice

Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMp(b)) and native wild-type (wt) empty capsids (MVMp(e)), determined and refined to 3.25 and 3.75 A resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 A resolution in this study. A comparison of the MVMp(b) and MVMp(e) capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the "shoulder" of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.     

Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing

Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.     

Characterization of two chemotypes of <Emphasis Type="Italic">Pinus pinaster</Emphasis> by their terpene and acid patterns in needles

The existence of two chemotypes of Pinus pinaster, on the basis of the chemical composition of the resin acids in their needles, is known. An investigation was performed on 54 samples of needles of Spanish Pinus pinaster to study the differences between these chemotypes on the basis of monoterpene, sesquiterpene, neutral diterpene, fatty acid, and resin acid composition. One-hundred and twelve compounds were identified by GC–FID and GC–MS. Statistical analysis of the results established the existence of two groups or chemotypes, in the ratio of 5:1. In one chemotype, total acid compounds were more abundant than neutral compounds, whereas in the other the concentrations of both neutral and acid compounds were similar. Distinction of the chemotypes was based on the presence/absence of a sesquiterpene (germacrene d-4-ol acetate), neutral diterpenes (8(14),13(15)-abietadiene, anticopalol, an isomer of anticopalol, and pimarol), fatty acids (10-octadecenoic, 14-hydroxy-10-octadecenoic, and 13-hydroxy-9-octadenoic acids and an unidentified fatty acid), and resin acids (levopimaric + palustric, eperuic, and anticopalic acids, and three isomers of anticopalic acid); and on the different relative percentages of other compounds of these types. This study gives a wide view of the composition of the needles of Pinus pinaster, improving the differentiation of chemotypes on the basis of terpene and acid composition.     

Human aging brain disorders: Role of antioxidant enzymes

In order to investigate the role of two free radical detoxificant enzymes in patients with aging brain disorders, superoxide dismutase (SOD) and catalase (CAT) activities have been measured in blood from male and female human patients of different ages with several types of aging brain disorders. When compared with activities in the normal population, we have detected: 1) SOD and CAT activities are decreased in patients with Parkinson disease. 2) SOD activity seems to be normal and CAT activity is decreased in patients with dementia. 3) In the patients with stroke, SOD activity is normal, while CAT activity is decreased. SOD activity was measured in red blood cells using the Minami and Yoshikawa method. CAT activity was measured in hemolysates by the method of Aebi. We can conclude that SOD and CAT activities in patients with Parkinson disease are decreased.     

Immunolocalization of beclin 1, a bcl-2-binding,autophagy-related protein,in the human ovary: possible relation to life span of corpus luteum

Ovarian tissue homeostasis is maintained by highly regulated cyclic phases of cell proliferation/differentiation and programmed cell death. Compelling evidence indicates that both apoptotic and autophagic types of programmed cell death are involved in the regression of the corpus luteum (CL) in primate species. Beclin 1 is an autophagy-related protein that is involved in the inter-relationships between apoptosis and autophagy, through interaction with the anti-apoptotic protein bcl-2. We studied the presence and expression pattern of beclin 1 in the adult human ovary. In ovarian follicles, beclin 1 immunostaining was found in the theca layer, whereas granulosa cells were negative. After ovulation, beclin 1 immunostaining was present in both theca-lutein and granulosa-lutein areas. The expression of beclin 1 in granulosa-lutein cells was related to the functional and structural status of the CL, being strong at the early and mid luteal phases, barely detectable at the late luteal phase, and absent in granulosa-lutein cells in subsequent cycles. Our results indicated that beclin 1 expression was related to luteal cell survival rather than to cell death. Accordingly, persistent beclin 1 expression was found in granulosa-lutein cells under either physiological (i.e., CL of pregnancy) or pathological (irregularly regressing CL in climacteric women) conditions involving prolonged CL life span. Strong beclin 1 immunostaining was also found in ovarian androgen-producing cells (i.e., secondary interstitial and hilus cells). Our data thus suggest that beclin 1 plays important roles in the regulation of the life span of human CL and ovarian androgen-secreting cells, by maintaining autophagy at levels promoting cell survival rather than cell death. This study was subsidized by grant BFU 2005-01443 from the DGICYT (Spain).     

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n=58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.     

Conjugates of ferrocene with biological compounds. Coordination to gold complexes and antitumoral properties

Several bioconjugates of ferrocene with biological compounds such as aminoacid esters and related species have been prepared by reaction of chlorocarbonyl ferrocene with the corresponding amino acid ester (histidine methyl ester, tryptophan methyl ester, methionine methyl ester and lysine ethyl ester) or histamine or prolinamide in the presence of NEt₃. The reaction of the tryptophan or prolinamide ferrocene conjugates with [Au(acac)(PR₃)] (acac = acetylacetonate) results in the substitution of the proton of the cyclic NH groups by the fragment AuPR₃⁺ affording the complexes [Au(FcCO-tryptophan-OMe)(PR₃)] or [Au(FcCO-prolinamide)(PR₃)] (Fc = ferrocenyl group). The reaction of FcCO-Met-OMe with [Au(OTf)(PR₃)] (OTF = trifluoromethysulfonate) or [Au(C₆F₅)₃(OEt₂)] yields the gold(I) or gold(III) derivatives [Au(FcCO-Met-OMe)(PR₃)]OTf or [Au(C₆F₅)₃(FcCO-Met-OMe)], respectively. Cytotoxicity studies towards several cancer lines such as MCF-7, HeLa or NIE-115 have been performed. The ferrocene bioconjugates show no activity whereas the gold complexes exhibit antiproliferative effect. Preliminary studies of interaction of compounds with cells were carried out with the goal of increasing our knowledge on the mechanism of action of these potential drugs.     

The Role of Polymerase Chain Reaction of High-Risk Human Papilloma Virus in the Screening of High-Grade Squamous Intraepithelial Lesions in the Anal Mucosa of Human Immunodeficiency Virus-Positive Males Having Sex with Males

Objectives

To evaluate the advantages of cytology and PCR of high-risk human papilloma virus (PCR HR-HPV) infection in biopsy-derived diagnosis of high-grade squamous intraepithelial lesions (HSIL = AIN2/AIN3) in HIV-positive men having sex with men (MSM).

Methods

This is a single-centered study conducted between May 2010 and May 2014 in patients (n = 201, mean age 37 years) recruited from our outpatient clinic. Samples of anal canal mucosa were taken into liquid medium for PCR HPV analysis and for cytology. Anoscopy was performed for histology evaluation.

Results

Anoscopy showed 33.8% were normal, 47.8% low-grade squamous intraepithelial lesions (LSIL), and 18.4% HSIL; 80.2% had HR-HPV. PCR of HR-HPV had greater sensitivity than did cytology (88.8% vs. 75.7%) in HSIL screening, with similar positive (PPV) and negative predictive value (NPV) of 20.3 vs. 22.9 and 89.7 vs. 88.1, respectively. Combining both tests increased the sensitivity and NPV of HSIL diagnosis to 100%. Correlation of cytology vs. histology was, generally, very low and PCR of HR-HPV vs. histology was non-existent (<0.2) or low (<0.4). Area under the receiver operating characteristics (AUROC) curve analysis of cytology and PCR HR-HPV for the diagnosis of HSIL was poor (<0.6). Multivariate regression analysis showed protective factors against HSIL were: viral suppression (OR: 0.312; 95%CI: 0.099-0.984), and/or syphilis infection (OR: 0.193; 95%CI: 0.045-0.827). HSIL risk was associated with HPV-68 genotype (OR: 20.1; 95%CI: 2.04-197.82).

Conclusions

When cytology and PCR HR-HPV findings are normal, the diagnosis of pre-malignant HSIL can be reliably ruled-out in HIV-positive patients. HPV suppression with treatment protects against the appearance of HSIL.