Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K_ATP) channels. K_ATP channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K_ATP channel subunit were compared with littermate controls. Evaluation of K_ATP channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80–85% reduction in cardiac K_ATP channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K_ATP channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.     

Identification of the motifs and amino acids in aggrecan G1 and G2 domains involved in product secretion

Members of the large aggregating chondroitin sulfate proteoglycans are characterized by an N-terminal fragment known as G1 domain, which is composed of an immunoglobulin (IgG)-like motif and two tandem repeats (TR). Previous studies have indicated that the expressed product of aggrecan G1 domain was not secreted. Here we demonstrated that the inability of G1 secretion was associated with the tandem repeats but not the IgG-like motif, and specifically with TR1 of aggrecan. We also demonstrated that the G2 domain, a domain unique to aggrecan, had a similar effect on product secretion. The sequence of TR1 of G1 is highly conserved across species, which suggested similar functions played by these motifs. In a yeast two-hybrid assay, TR1 interacted with the calcium homeostasis endoplasmic reticulum protein. Deletion/mutation experiments indicated that the N-terminal fragment of TR1, in particular, the amino acids H(2)R(4) of this motif were key to its effect on product secretion. However, the N-terminal 55 amino acids were required to exert this function. Taken together, our study suggests a possible molecular mechanism for the function of the tandem repeats in product processing.     

Microbial diversity of biofilms in dental unit water systems

We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods. The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH). Most bacteria present in the biofilm were viable. FISH detected the beta and gamma, but not the alpha, subclasses of Proteobacteria: In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing. Only 16S identified all 55 isolates, which represented 13 genera. The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera Afipia (28%) and Sphingomonas (16%). The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA. This method revealed 40 genera: the most common ones included Leptospira (20%), Sphingomonas (14%), Bacillus (7%), Escherichia (6%), Geobacter (5%), and Pseudomonas (5%). Some of these organisms may be opportunistic pathogens. Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms. The results also reflect the limitations of culture-based techniques to detect and identify bacteria. Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed. Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms.     

Biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria

As an energy carrier, hydrogen gas is a promising substitute to carbonaceous fuels owing to its superb conversion efficiency, non-polluting nature, and high energy content. At present, hydrogen is predominately synthesized via chemical reformation of fossil fuels. While various biological methods have been extensively explored, none of them is justified as economically feasible. A sustainable platform for biological production of hydrogen will certainly impact the biofuel market. Among a selection of biological systems, algae and cyanobacteria have garnered major interests as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical systems. This article reviews recent advances of biochemical, bioprocess, and genetic engineering strategies in circumventing technological limitations to hopefully improve the applicative potential of these photosynthetic hydrogen production systems.     

A unique error signature for human DNA polymerase nu

Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.     

Role of MGMT in protecting against cyclophosphamide-induced toxicity in cells and animals

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects cells from the biological consequences of alkylating agents by removing alkyl groups from the O(6)-position of guanine. Cyclophosphamide and ifosfamide are oxazaphosphorines used clinically to treat a wide variety of cancers; however, the role of MGMT in recognizing DNA damage induced by these agents is unclear. In vitro evidence suggests that MGMT may protect against the urotoxic oxazaphosphorine metabolite, acrolein. Here, we demonstrate that Chinese hamster ovary cells transfected with MGMT are protected against cytotoxicity following treatment with chloroacetaldehyde (CAA), a neuro- and nephrotoxic metabolite of cyclophosphamide and ifosfamide. The mechanism by which MGMT recognizes damage induced by acrolein and CAA is unknown. CHO cells expressing a mutant form of MGMT (MGMT(R128A)), known to have >1000-fold less repair activity towards alkylated DNA while maintaining full active site transferase activity towards low molecular weight substrates, exhibited equivalent CAA- and acrolein-induced cytotoxicity to that of CHO cells transfected with plasmid control. These results imply that direct reaction of acrolein or CAA with the active site cysteine residue of MGMT, i.e. scavenging, is unlikely a mechanism to explain MGMT protection from CAA and acrolein-induced toxicity. In vivo, no difference was detected between Mgmt-/- and Mgmt+/+ mice in the lethal effects of cyclophosphamide. While MGMT may be important at the cellular level, mice deficient in MGMT are not significantly more susceptible to cyclophosphamide, acrolein or CAA. Thus, our data does not support targeting MGMT to improve oxazaphosphorine therapy.     

Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation

A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.     

Targeted deletion of LPA5 identifies novel roles for lysophosphatidic acid signaling in development of neuropathic pain

Lysophosphatidic acid (LPA) is a bioactive lipid that serves as an extracellular signaling molecule acting through cognate G protein-coupled receptors designated LPA(1-6) that mediate a wide range of both normal and pathological effects. Previously, LPA(1), a G(αi)-coupled receptor (which also couples to other G(α) proteins) to reduce cAMP, was shown to be essential for the initiation of neuropathic pain in the partial sciatic nerve ligation (PSNL) mouse model. Subsequent gene expression studies identified LPA(5), a G(α12/13)- and G(q)-coupled receptor that increases cAMP, in a subset of dorsal root ganglion neurons and also within neurons of the spinal cord dorsal horn in a pattern complementing, yet distinct from LPA(1), suggesting its possible involvement in neuropathic pain. We therefore generated an Lpar5 null mutant by targeted deletion followed by PSNL challenge. Homozygous null mutants did not show obvious base-line phenotypic defects. However, following PSNL, LPA(5)-deficient mice were protected from developing neuropathic pain. They also showed reduced phosphorylated cAMP response element-binding protein expression within neurons of the dorsal horn despite continued up-regulation of the characteristic pain-related markers Caα(2)δ(1) and glial fibrillary acidic protein, results that were distinct from those previously observed for LPA(1) deletion. These data expand the influences of LPA signaling in neuropathic pain through a second LPA receptor subtype, LPA(5), involving a mechanistically distinct downstream signaling pathway compared with LPA(1).     

Biochemical characterization of human enteropeptidase light chain

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.