The protective role of Coenzyme Q10 in metallothionein-3 expression in liver and kidney upon rats’ exposure to lead acetate

Molecular Biology Reports - Metallothionein-3 (MT3) is an antioxidant protein that alters after exposure to heavy metals. In this study, we investigated the hepatic and renal expression of MT3 gene...     

Photobiomodulation of a flowable matrix in a human skin ex vivo model demonstrates energy‐based enhancement of engraftment integration and remodeling

The use of dermal substitutes to treat skin defects such as ulcers has shown promising results, suggesting a potential role for skin substitutes for treating acute and chronic wounds. One of the main drawbacks with the use of dermal substitutes is the length of time from engraftment to graft take, plus the risk of contamination and failure due to this prolonged integration. Therefore, the use of adjuvant energy‐based therapeutic modalities to augment and accelerate the rate of biointegration by dermal substitute engraftments is a desirable outcome. The photobiomodulation (PBM) therapy modulates the repair process, by stimulating cellular proliferation and angiogenesis. Here, we evaluated the effect of PBM on a collagen‐glycosaminoglycan flowable wound matrix (FWM) in an ex vivo human skin wound model. PBM resulted in accelerated rate of re‐epithelialization and organization of matrix as seen by structural arrangement of collagen fibers, and a subsequent increased expression of alpha‐smooth muscle actin (α‐SMA) and vascular endothelial growth factor A (VEGF‐A) leading to an overall improved healing process. The use of PBM promoted a beneficial effect on the rate of integration and healing of FWM. We therefore propose that the adjuvant use of PBM may have utility in enhancing engraftment and tissue repair and be of value in clinical practice.      

Left Ventricular Hypertrophy in Rhesus Macaques (Macaca mulatta) at the California National Primate Research Center (1992–2014)

Necropsy records and associated clinical histories from the rhesus macaque colony at the California National Primate Research Center were reviewed to identify mortality related to cardiac abnormalities involving left ventricular hypertrophy (LVH). Over a 21-y period, 162 cases (female, 90; male, 72) of idiopathic LVH were identified. Macaques presented to necropsy with prominent concentric hypertrophy of the left ventricle associated with striking reduction of the ventricular lumen. Among all LVH cases, 74 macaques (female, 39; male, 35), mostly young adults, presented for spontaneous (sudden) death; more than 50% of these 74 cases were associated with a recent history of sedation or intraspecific aggression. The risk of sudden death in the 6- to 9-y-old age group was significantly higher in male macaques. Subtle histologic cardiac lesions included karyomegaly and increased cardiac myocyte diameter. Pedigree analyses based on rhesus macaque LVH probands suggested a strong genetic predisposition for the condition. In humans, hypertrophic cardiomyopathy (HCM) is defined by the presence of unexplained left ventricular hypertrophy, associated with diverse clinical outcomes ranging from asymptomatic disease to sudden death. Although the overall risk of disease complications such as sudden death, end-stage heart failure, and stroke is low (1% to 2%) in patients with HCM, the absolute risk can vary dramatically. Prima facie comparison of HCM and LVH suggest that further study may allow the development of spontaneously occurring LVH in rhesus macaques as a useful model of HCM, to better understand the pathogenesis of this remarkably heterogeneous disease.Abbreviations: HCM, hypertrophic cardiomyopathy; LVH, left ventricular hypertrophyNaturally occurring hypertrophic cardiomyopathy (HCM) has been recognized in a variety of species including pigs,¹³ cats^18,32 and humans. HCM emerged as an accepted clinical entity in humans during the late 1950s, with the publication of 2 key papers.^5,46 Since then, a vast, complex, and sometimes contradictory body of research has developed around this clinically, phenotypically, and genotypically heterogeneous disease. HCM is defined as left ventricular hypertrophy without chamber dilation in the absence of either a systemic or other cardiac disease that would result in pressure overload and compensatory hypertrophy.^22,39 The primary physiologic abnormality in HCM is reduced stroke volume due to impaired diastolic filling, which is secondary to reduced chamber size and impaired relaxation of the left ventricular myocardium during diastole; this impaired relaxation is due to the reduced compliance or increased stiffness of the hypertrophied left ventricle.⁴⁹Phenotypically, hearts affected by HCM exhibit a wide variety of changes summarized as left ventricular hypertrophy, which may be symmetric or asymmetric and which results in reduced left ventricular lumen volume, reduced stroke volume, and typically increased ejection fraction. These changes can be accompanied by valvular changes, some of which can be obstructive. Small intramural coronary arteries may have thickened walls and narrowed lumens due to proliferation of smooth muscle cells and collagen.³⁰ Histologic changes in the left ventricle associated with—but not prerequisite for—HCM include myocyte hypertrophy and disarray, nuclear atypia, and expansion of the interstitial collagen compartment.^14,22In the 1980s, growing recognition that HCM was familial directed efforts toward identifying a genetic defect. Primary HCM in humans has been identified as an autosomal dominant disease with variable penetrance and a remarkable diversity in clinical presentation and disease course.^27,40 The first mutation associated with HCM was a missense mutation in the gene encoding the β-myosin heavy chain.¹¹ More than 2 decades of subsequent investigation has demonstrated the extensive heterogeneity of the HCM phenotype, with at least 11 causative genes and more than 1400 mutations identified. These genes primarily encode thick and thin myofilament proteins of the sarcomere or Z disc (sarcomere structural proteins). The majority of mutations occur in 3 genes—β-myosin heavy chain, myosin-binding protein C, and troponin T—whereas other genes, including troponin I, α-tropomyosin and α-actin, account for a smaller proportion of patients.²² Mutations in several additional sarcomere and calcium-handling genes have been proposed but with less evidence to support pathogenicity. At present, the precise mutation does not alter management. However, adverse outcomes (sudden death, stroke, progressive symptoms) are more prominent in patients with sarcomere mutations than in those without an identifiable mutation.³⁷In other animal species, naturally occurring familial HCM has been best characterized in cats. Maine coon cats¹⁸ and ragdoll cats³² develop HCM that is strongly similar to the human disease in clinical presentation and histopathology. Similar to humans, HCM in cats is inherited as an autosomal dominant trait, with the responsible gene, the cardiac myosin-binding protein C gene, recently identified.^32,33 In addition, a genetically manipulated model has been developed in rabbits,²¹ as well as a vast array of mouse models.^1,7,45The purposes of this report were: 1) to identify cases of LVH diagnosed at necropsy in the rhesus macaque colony at the California National Primate Research Center since 1992; 2) to provide a preliminary pathologic characterization of these cases; and 3) to compare and contrast rhesus LVH and human HCM to assess the extent to which LVH might be developed as a model of HCM in a species closely related to humans.     

Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates

Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.     

cDNA sequence encoding a Plasmodium falciparum protein associated with knobs and localization of the protein to electron-dense regions in membranes of infected erythrocytes.

Plasmodium falciparum modifies the host erythrocyte's plasma membrane by the formation of electron-dense structures called knobs. We have produced monoclonal antibodies (McAbs) which specifically bind to the knobs in immunoelectron microscopic experiments with thin sections of parasitized erythrocytes. However, the McAbs fail to bind to the surface of live parasitized erythrocytes. Immunoblotting experiments with these McAbs show the antigen is localized to the erythrocyte plasma membrane. The antigen with which the McAbs react varies in mol. wt from 80 to 95 kd in different knob-producing isolates of P. falciparum and is absent in knobless variants. The McAbs react with the expressed product of a P. falciparum cDNA clone, thus demonstrating that the clone encodes part of this knob-associated protein. The sequence of the cDNA fragment partially overlaps a published cDNA sequence reported to encode the amino-terminal portion of the knob protein, and extends the predicted open reading frame by 190 amino acids. The carboxyl-terminal portion of the predicted amino acid sequence contains a highly charged stretch of approximately 100 amino acid residues. We suggest that this unusual, highly charged region participates in intermolecular salt bridging leading to dense packing of these molecules. This would create the electron-dense regions observed by electron microscopy and might also explain the insolubility of the knob-associated protein in the absence of strong ionic detergents or chaotropic agents.     

In Vitro Study of Novel Collagenase (XIAFLEX?) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

Dupuytren''s disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH) - Xiaflex® - is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.     

Isolation and Characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium.

Mutations in the ack (acetate kinase) and pta (phosphotransacetylase) genes in Salmonella typhimurium were characterized and determined to be analogous to those of previously described Escherichia coli mutants. We established that in both bacterial species these genes were cotransducible with the neighboring histidine transport operon and were distally located relative to purF. pta mutants were sensitive to the dye alizarin yellow and were unable to grow on medium containing inositol as a carbon source. We selected mutants of both species with deletions covering both the ack and the pta genes; some deletions extended into the histidine transport operon.