Bone fragility after spinal cord injury: reductions in stiffness and bone mineral at the distal femur and proximal tibia as a function of time.

Computed tomography and finite element modeling were used to assess bone structure at the knee as a function of time after spinal cord injury. Analyzed regions experienced degradation in stiffness, mineral density, and content. Changes were well described as an exponential decay over time, reaching a steady state 3.5 years after injury. INTRODUCTION: Spinal cord injury (SCI) is associated with bone fragility and an increased risk of fracture around the knee. The purpose of this study was to investigate bone stiffness and mineral content at the distal femur and proximal tibia, using finite element (FE) and computed tomography (CT) measures. A cross-sectional design was used to compare differences between non-ambulatory individuals with SCI as a function of time after injury (0-50 years). METHODS: CT scans of the knee were obtained from 101 individuals who experienced an SCI 30 days to 50 years prior to participation. Subject-specific FE models were used to estimate stiffness under axial compression and torsional loading, and CT data was analyzed to assess volumetric bone mineral density (vBMD) and bone mineral content (BMC) for integral, cortical, and trabecular compartments of the epiphyseal, metaphyseal, and diaphyseal regions of the distal femur and proximal tibia. RESULTS: Bone degradation was well described as an exponential decay over time (R2 = 0.33-0.83), reaching steady-state levels within 3.6 years of SCI. Individuals at a steady state had 40 to 85% lower FE-derived bone stiffness and robust decreases in CT mineral measures, compared to individuals who were recently injured (t ≤ 47 days). Temporal and spatial patterns of bone loss were similar between the distal femur and proximal tibia. CONCLUSIONS: After SCI, individuals experienced rapid and profound reductions in bone stiffness and bone mineral at the knee. FE models predicted similar reductions to axial and torsional stiffness, suggesting that both failure modes may be clinically relevant. Importantly, CT-derived measures of bone mineral alone underpredicted the impacts of SCI, compared to FE-derived measures of stiffness. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01225055, NCT02325414).

Structure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

A gene encoding laccase has been isolated from a genomic library of the white-rot basidiomycete Ceriporiopsis subvermispora constructed in Lambda GEM-11. This gene (Cs-lcs1) contains an open reading frame of 2215 bp, encoding a mature protein of 499 amino acids with a 21-residue signal peptide. The protein sequence exhibits between 63 and 68% identity with laccases from other basidiomycetes and shares with all of them 10 conserved histidines and one cysteine involved in the coordination of copper atoms at the active site of the enzyme. The gene possesses 11 introns, with splicing junctions and internal lariat formation sites adhering to the GT-AG and CTRAY rules, respectively. The upstream region of Cs-lcs1 contains a TATA box, two CAAT sites, five putative metal response elements and a ACE1 element. In agreement with the presence of the latter element, transcription of Cs-lcs1 is activated by copper and silver, as shown by Northern blot and reverse transcription followed by DNA amplification analyses. Based on Southern blot analysis, Cs-lcs1 appears to be the only gene encoding laccase in C. subvermispora.

Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

Three new genes (Cs-mnp2A, Cs-mnp2B and Cs-mnp3) coding for manganese-dependent peroxidase (MnP) have been identified in the white-rot basidiomycete Ceriporiopsis subvermispora. The mature proteins contain 366 (MnP2A and MnP2B) and 364 (MnP3) amino acids, which are preceded by leader sequences of 21 and 24 amino acids, respectively. Cs-mnp2A and Cs-mnp2B appear to be alleles, since the corresponding protein sequences differ in only five residues. The upstream region of Cs-mnp2B contains a TATA box, AP-1 and AP-2 sites, as well as sites for transcription regulation by metals (two), cAMP (two) and xenobiotics (one). Some of these elements are also found in the regulatory region of Cs-MnP3. Transcription of Cs-mnp2A and Cs-mnp2B, but not that of Cs-mnp3, is activated by manganese.

Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora.

A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I-P-X-P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5kb contained the complete sequence of the Cs-mnp1 gene, including 162bp and 770bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT-AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9kb was amplified using inverse PCR. A putative TATAA element was identified 5' of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.

Native and chemically modified porin channels from Salmonella typhi Ty2 in planar lipid bilayers.

Native porins, from Salmonella typhi Ty2 outer membrane, and porins alkylated with pyridoxal phosphate (Plp) were studied in planar lipid bilayers. The conductance of bilayers exposed to native or chemically modified porins increases in discrete jumps. Conductance histograms for native porins displayed two major peaks at 1.7 and 6.7 nS (in 0.5 M KCl). On the other hand, Plp-treated porins exhibited a single major peak at 1 nS. The relation between bilayer conductance and native porin concentration was linear. However, this relation became logarithmic in the presence of modified porins. The results support the notion that alkaline reduction of S. typhi Ty2 porins with Plp dissociates porin channel trimers in a reversible fashion.

Oxidation reactions catalyzed by manganese peroxidase isoenzymes from Ceriporiopsis subvermispora.

A total of 11 manganese peroxidase isoenzymes (MnP1-MnP11) with isoelectric points (pIs) in the range of 4.58-3.20 were isolated from liquid- and solid-state cultures of the basidiomycete Ceriporiopsis subvermispora. In the presence of hydrogen peroxide, these isoenzymes showed different requirements for Mn(II) in the oxidation of vanillylacetone, o-dianisidine, p-anisidine and ABTS, whereas oxidation of guaiacol by any isoenzyme did not take place when this metal was omitted. Km values for o-dianisidine and p-anisidine in the absence of Mn(II) are in the range of 0.5-1.0 mM and 4.5-42.0 mM, respectively. Oxalate and citrate, but not tartrate, accelerate the oxidation of o-dianisidine, both in the presence and in the absence of Mn(II). MnPs from this fungus are able to oxidize kojic acid without externally added hydrogen peroxide, indicating that they can also act as oxidases. In this reaction, however, the requirement for Mn(II) is absolute.

Isolation and characterization of homokaryotic strains from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

Genetic analyses of the lignin-degrading fungus Ceriporiopsis subvermispora is complicated by a dikaryotic nuclear condition and the absence of spore forms. Previous investigations had identified a family of closely related sequences encoding manganese peroxidase (MnP), but the relationship between genes and allelic variants could not be experimentally established. Addressing this issue, homokaryotic derivatives of C. subvermipora strain FP105752 were isolated from regenerated protoplasts. Designated CsA and CsB, their homokaryotic nature was established by polymerase chain reaction amplification and sequence analysis of the allelic variants of three MnP genes. Isoelectrofocusing revealed fewer MnP isoenzymes in filtrates of homokaryon cultures relative to the parental strain. The homokaryotic strains will simplify genetic analyses, particularly the identification of new genes.

Association of the intronic polymorphism rs891512 (G24943A) of the endothelial nitric oxide synthase gene with hypertension in Chilean type 2 diabetes patients.

We investigated two single nucleotide polymorphisms of the NOS3 gene in type 2 diabetic patients (n=93) and healthy non-diabetic controls (n=76) and their relationship with smoking habits, body mass index, hypertension and dyslipidemia. Results showed that eNOS polymorphism rs891512 (G24943A) is associated with hypertension in Chilean individuals (p<0.05).

Finasteride: mitos y verdades / Finasteride: myths and truths

La alopecia androgénica consiste en un patrón específico de caída de pelo. Es más común en el sexo masculino. Antes de la caída total del pelo, ocurre una transformación del pelo grueso, pigmentado y terminal en vellos pequeños, hipopigmentados y finos, fenómeno dado por aumento en la afinidad de los receptores del pelo a andrógenos. La dihidrotestosterona es un andrógeno con una afinidad 5 veces mayor por los receptores en el pelo del cuero cabelludo. La enzima 5 a-reductasa cataliza la transformación de testosterona a dihidrotestosterona. El finasteride es un bloqueador selectivo de la 5 a -reductasa tipo 2 utilizado como tratamiento para la alopecia de patrón andrógeno. Existen numerosas controversias respecto a la eficacia y a los efectos no deseados que pudiera causar el finasteride; éste artículo pretende dilucidar aquellos más relevantes.

Androgenetic alopecia is characterized by progressive, patterned hair loss from the scalp. The transition of terminal hair into vellus hairs is characteristic, and attributed to an increased affinity of hair androgen receptors to circulating androgens. It is considered to be a genetically, hereditary condition. Among androgens, dihydrotestosterone (DHT) has approximately five fold greater affinity for the androgen receptor compared to testosterone. DHT is a result of peripheral conversion of testosterone, a reaction catalyzed by the enzyme 5 a-reductase. Finasteride, a type 2-selective inhibitor of 5 a -reductase, was first used to treat benign prostate hyperplasia and later to treat androgenetic alopecia, with good outcomes. Many controversies arise related to the efficacy and safety of finasteride; the objective of this article is to elucidate the most relevant information.

Cáncer de piel no-melanoma / Non-melanoma skin cancer

El cáncer de piel no-melanoma incluye principalmente las neoplasias queratinocíticas (carcinoma basocelulary espinocelular) y tumores de menor frecuencia tales como: linfomas cutáneos, carcinoma de células de Merkel, sarcoma de Kaposi, angiosarcomas, enfermedad de Paget, e histiocistomas malignos entre otros. En este artículo revisaremos la epidemiología, patogénesis, clínica, histopatología, diagnóstico y modalidades terapéuticas de los dos principales cánceres de la piel no melanoma: basocelular y espinocelular.

The non-melanoma skin cancer refers mainly to the queratinocitic neoplasias (basal cell carcinoma and spinouscell carcinoma) and less frequently tumours like: lymphomas, Merkel cell carcinoma, Kaposi´s Sarcoma, angio sarcomas, Paget disease and malignant hystiocitomas. In this article we will review the epidemiology, pathogenesis, clinical picture, histopathology, diagnosis and treatment of the two more frequent non-melanoma skin cancers: basal cell carcinoma and squamous cell carcinoma.

Alteration in the electrophoretic mobility of OmpC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Studies on Salmonella typhi and Salmonella typhimurium outer membrane proteins have shown that the relative position of OmpC porin in sodium dodecyl sulfate.polyacrylamide gel electrophoresis undergoes an important shift when the concentration of ammonium persulfate in the running gel is increased from 6 to 12 mM. The apparent molecular mass at these concentrations was estimated to be 34 and 40 kDa, respectively. Under similar electrophoretic conditions the apparent molecular mass estimated for OmpF was 37.6 and 38.2 kDa. Therefore, OmpC moves from a leading position to a position behind OmpF. For Escherichia coli OmpC the shift observed is less pronounced than that occurring in Salmonellae.

The hemolytic effect of Salmonella typhi Ty 2 porins.

Two outer membrane proteins of Salmonella typhi Ty 2 were extensively co-purified. According to their migration in dodecylsulfate/polyacrylamide gel electrophoresis and solubility characteristics, these proteins are homologous to the 35-kDa and 36-kDa porins found in Salmonella typhimurium. A porin homologous to the 34-kDa one has not been found in S. typhi Ty 2. A critical step in the purification of porins is heating at 100 degrees C in 2% sodium dodecyl sulfate before Sephadex gel filtration. The absence of detergent in aqueous suspensions enhances porin aggregation, these aggregations inducing human red cell lysis. Porins obtained by an alternative procedure consisting of heating at 60 degrees C instead of 100 degrees C were also hemolytic. Using nanomolar concentration of porins a strong influence of temperature on the hemolytic effect was observed. Porin-induced hemolysis was inhibited with anti-porin serum, as well as by a treatment with phenylglyoxal, which reacts with the arginine residues of proteins. The membrane-disrupting ability of porins aggregates might explain some pathogenic characteristics of gram-negative bacterial infections.

Clinical isolate of a porinless Salmonella typhi resistant to high levels of chloramphenicol.

We studied a clinical isolate of Salmonella typhi (strain 1895) characterized by resistance to 200 micrograms of chloramphenicol per ml despite the absence of chloramphenicol-inactivating activity. The outer membrane protein profile analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a deficiency of one of the major protein species which may serve as a porin for entry of chloramphenicol. When the strain was transformed with a plasmid encoding chloramphenicol acetyltransferase, chloramphenicol added to the culture was not inactivated, suggesting a drastic reduction of permeability towards the drug. Moreover, transformants bearing a plasmid coding for the Escherichia coli OmpF porin became considerably more susceptible to chloramphenicol (40 micrograms/ml). On the other hand, transformants carrying a plasmid encoding the Salmonella typhi ompC gene remained as resistant to the drug as the parental strain, even though they overexpressed OmpC. These findings indicate that the lack of OmpF plays a major role in the resistance to chloramphenicol in strain 1895.

Factors involved in specific transcription by mammalian RNA polymerase II. Role of factors IID and MLTF in transcription from the adenovirus major late and IVa2 promoters.

The role of the adenovirus major late upstream transcription factor (MLTF) in transcription from the adenovirus major late and the IVa2 promoters was studied. The transcription initiation site of the IVa2 promoter is located 210 nucleotides upstream from the CAP site of the major late promoter. Transcription from these two promoters occurs on different DNA strands. Thus, this divergent transcription suggests that the same factor could simultaneously regulate the expression of two different genes. This was investigated utilizing a reconstituted transcription system in vitro. The addition of MLTF to reaction mixtures containing the purified general transcription factors and the major late promoter resulted in a 10-12-fold stimulation of transcription. This stimulation was because of an increase of the stability of the preinitiation complex. MLTF allowed DNA template molecules to undergo multiple rounds of transcription. MLTF also stimulated transcription from the adenovirus-encoded IVa2 promoter. Surprisingly, reconstitution experiments indicated that transcription from the IVa2 promoter which does not have a TATA sequence required all the previously described general transcription factors, including TFIID, the TATA binding protein. The requirement for TFIID was demonstrated by reconstitution experiments as well as by oligonucleotide competition experiments. The implications of this observation are discussed.

Isoenzymes of manganese-dependent peroxidase and laccase produced by the lignin-degrading basidiomycete Ceriporiopsis subvermispora.

The white-rot basidiomycete Ceriporiopsis subvermispora produces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pI values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pI 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II), mainly the MnPs with higher pI values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermispora when growing on wood chips of Pinus radiata. Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pI values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pI values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pI values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium.

We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.

Lip-like genes in Phanerochaete sordida and Ceriporiopsis subvermispora, white rot fungi with no detectable lignin peroxidase activity.

Lignin peroxidase-like genes were PCR amplified from Phanerochaete sordida and Ceriporiopsis subvermispora, fungi lacking lignin peroxidase (LiP) activity. Amplification products were highly similar to previously described LiP genes. Using reverse transcription-coupled PCR a LiP-like cDNA clone was amplified from P. sordida RNA. In contrast, no evidence was obtained for transcription of C. subvermispora LiP genes.

Properties of laccase isoenzymes produced by the basidiomycete Ceriporiopsis subvermispora.

Laccase is one of the ligninolytic enzymes found in liquid cultures of the fungus Ceriporiopsis subvermispora in defined medium. As an approach to a clarification of the role of laccases during the attack on lignin by the fungus, the enzyme has been characterized further. The levels of this phenol oxidase increase 2-fold in the presence of p-anisidine and are severely affected when addition of either Mn(II) or Cu(II) ions to the medium is omitted. Isoelectrofocusing allowed the resolution of two laccase isoenzymes, with pIs of 3.65 and 3.59. In rich medium, laccase activity is 10-fold higher than in salt medium, and it is not affected by the external addition of p-anisidine or Mn(II). Four isoenzymes were detected in these cultures, with pIs between 3.76 and 3.60. In a wheat bran medium, four isoenzymes with pIs in the range 3.63-3.46, plus a fifth isoenzyme of high pI (4.82), were also identified. The absorption spectrum of a pool containing the four isoenzymes from rich medium shows a maximum at 600 nm, typical of laccase possessing a type I copper atom. The molecular mass of the isoenzyme with pI 3.60 is 79 kDa, as determined by SDS/PAGE. Upon treatment with endoglycosidase F, the molecular mass of this isoform decreases to 63 kDa, indicating a high degree of glycosylation. Substrate specificity studies conducted with the four isoenzymes from rich medium and a combination of isoenzymes from salt medium showed marked differences among them. The amino-terminal sequences (24 residues) of three isoenzymes isolated from rich medium were determined. Two of them are identical, whereas the third one differs from these in three amino acid residues. The consensus sequence reveals clear homology with laccases from other microorganisms.

Antibodies to porin antigens of Salmonella typhi induced during typhoid infection in humans.

Immunoglobulin G (IgG)- and IgM-specific antibody titers against Salmonella typhi Ty2 porins have been measured in 30 paired typhoid sera by enzyme-linked immunosorbent assay. These studies have found that IgG serum titers of acute and convalescent sera were 625 and 5,000 times higher, respectively than the control serum titers. The same typhoid sera were titrated with S. typhi Ty2 flagellin and S. typhi lipopolysaccharide. The titers against these antigens were considerably lower than those against the porins. The highest IgM-specific titer has also been found against porins in convalescent-phase sera. However, the largest increase in IgM-specific titer compared with the control group titer was obtained against flagellin during the acute phase of typhoid. The lowest increases in antibody titer were obtained with the IgM-specific anti-lipopolysaccharide in both types of sera. This may be because many normal individuals in endemic areas already have IgM titers against lipopolysaccharide. This study has provided good evidence that porins are excellent antigens and that IgG-specific antiporin titers may be of diagnostic value in typhoid infections in endemic areas.

Estudio de la sensibilidad y especificidad diagnóstica del microscopio confocal de reflectancia (MCR) en lesiones tumorales de la piel / Diagnostic sensitivity and specificity of the Reflectance Confocal Laser Microscope (RCLM) in tumors of the skin

Introducción: El Microscopio Láser Confocal de Reflectancia (MCR) es una técnica no invasiva, con resolución cuasi histológica, que permite la visualización de células y estructuras de la piel. Esta tecnología es especialmente útil en el diagnóstico de lesiones tumorales. Material y métodos: Se trata de un estudio retrospectivo que busca medir la sensibilidad y especificidad del MCR en el diagnóstico de lesiones tumorales de la piel. Luego de obtener imágenes con el MCR de las lesiones, éstas se resecaron y se enviaron a estudio histopatológico, observando la correlación entre ambas técnicas. Resultados: Encontramos un 98 por ciento de concordancia entre el diagnóstico por MCR y el diagnóstico histopatológico para el total de tumores. El MCR mostró una sensibilidad y especificidad de 100 por ciento paramelanomas y sensibilidad de 93 por ciento y especificidad de 100 por ciento para carcinoma basocelular. En el caso de nevus atípico, la sensibilidad fue de 100 por ciento y la especificidad de 97 por ciento. Sólo dos lesiones que en la histopatología demostraron ser carcinomas basocelulares superficial y nodular fueron confundidas conqueratosis liquenoide y nevus atípico respectivamente. Discusión: Este estudio demostró una alta concordancia entre el MCR y la histopatología en tumores malignos. Sin duda que el MCR es y seguirá siendo un pilar fundamental en el diagnóstico de las lesiones tumorales de la piel, así como en la investigación y seguimiento in vivo de diversas lesiones de la piel.

Introduction: The Reflectance Confocal Laser Microscope (RCLM) is a new non-invasive technology, with quasi-histological resolution, which allows visualization of cells and skin structures and is particularly useful in the diagnosis of skin tumors. Material and methods: This is a retrospective study aimed to determine the diagnostic sensitivity and specificity of the RCLM in tumors of the skin using conventional histology as the gold standard for diagnosis. The images were obtained from patients with tumors of the skin using the RCLM, which were subsequently excised and sent to histopathological analysis. Results: We found a 98 percent concordance between the previous diagnosis by RCLM and pathology results. Diagnosis by confocal microscopy showed a sensitivity and specificity of 100 percent for melanomas, 93 percent sensitivity and 100 percent specificity for basal cell carcinoma and a sensitivity of 100 percent and specificity of 97 percent for atypical nevi. Only 2 lesions on histopathology proved to be superficial and nodular basal cell carcinomas were confused with lichenoid keratosis and atypical nevi, respectively. Discussion: We believe that RCLM is and will remain a really useful tool in the diagnosis of tumours of the skin as well as in research and in vivo monitoring of various types of skin lesion.