Distinct Isozymes Research Articles

Inactivation of polyphenol oxidase and peroxidase in pineapple juice during continuous ohmic heating and modeling of inactivation kinetics during isothermal holding

AbstractContinuous ohmic heating (COH) is a novel thermal technology for fruit juice processing and preservation. The present work demonstrated the effect of COH parameters like electric field strength (EFS: 30–40 V/cm), isothermal holding (0–60 s), and temperature (70–90°C) on the inactivation of polyphenol oxidase (PPO) and peroxidase (POD) in standardized pineapple juice. Statistical parameters and Akaike information criteria were used to compare kinetic models like the first order, distinct isozymes, Weibull distribution, sigmoidal logistic, and fractional conversion for PPO and POD inactivation. The findings revealed that the COH parameters significantly affected the activities of both enzymes. A maximum inactivation of 68.2% of PPO and 82.2% of POD was observed at 90°C, 60 s, and 40 V/cm. The Weibull distribution model, compared to other models, was found to describe the inactivation kinetics better for both PPO (R2 > 0.99; RMSE < 0.0101) and POD (R2 > 0.98; RMSE < 0.042). The accuracy factor (Af) and bias factor (Bf) for both PPO (Af = 1.003–1.010; Bf = 1.000–1.004) and POD (Af = 1.007–1.072; Bf = 0.992–1.009) were closer to simulation line suggesting the accuracy of the Weibull model in predicting the enzyme inactivation. Also, Akaike increment (∆i < 2) substantially supported the Weibull model. The shape factor (n < 1) explained the tailing phenomenon of the enzymes. The PPO's scale factor (δ values) was higher than POD, suggesting higher thermal stability of PPO than POD. Thus, the Weibull model is a good tool for predicting the enzyme inactivation in COH‐treated standardized pineapple juice.Practical ApplicationsContinuous ohmic heating (COH) is a novel electro‐thermal technology that heats the fruit juice quickly, uniformly, and volumetrically. Generally, for inactivating spoilage enzymes like polyphenol oxidase and peroxidase during pineapple juice processing in an industrial set‐up, a conventional method of conduction and convection mode of heat transfer is used. This results in a high‐temperature gradient within the juice matrix, causing a problem of over‐ and under‐processing of juice. Ohmic heating is a potential and efficient way of inactivating these spoilage enzymes to a desired level in a shorter processing time. It can be used to preserve juice by extending its shelf life. COH can reduce the total heat damage to the juice by maintaining its flavor and nutrient compounds. Overall, ohmic heating is a versatile and effective technology for the continuous processing of fruit juice for improved product quality and production efficiency.

Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins.

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.

Biological and Pharmacological Properties of Carbon Monoxide: A General Overview

Carbon monoxide (CO) is one of the most common causes of inhalation poisoning worldwide. However, it is also well known that CO is produced endogenously in the heme degradation reaction catalyzed by heme oxygenase (HO) enzymes. HO catalyzes the degradation of heme to equimolar quantities of CO, iron ions (Fe2+), and biliverdin. Three oxygen molecules (O2) and the electrons provided by NADPH-dependent cytochrome P450 reductase are used in the reaction. HO enzymes comprise three distinct isozymes: the inducible form, heme oxygenase-1 (HO-1); the constitutively expressed isozyme, heme oxygenase-2 (HO-2); and heme oxygenase-3 (HO-3), which is ubiquitously expressed but possesses low catalytic activity. According to some authors, HO-3 is rather a pseudogene originating from the HO-2 transcript, and it has only been identified in rats. Therefore, cellular HO activity is provided by two major isoforms—the inducible HO-1 and the constitutively expressed HO-2. For many years, endogenously generated CO was treated as a by-product of metabolism without any serious physiological or biochemical significance, while exogenous CO was considered only as an extremely toxic gas with lethal effects. Research in recent years has proven that endogenous and exogenous CO (which may be surprising, given public perceptions) acts not only as an agent that affects many intracellular pathways, but also as a therapeutic molecule. Hence, the modulation of the HO/CO system may be one option for a potential therapeutic strategy. Another option is the administration of CO by exogenous inhalation. As alternatives to gas administration, compounds known as CO-releasing molecules (CORMs) can be administered, since they can safely release CO in the body. The aim of this article is to provide a brief overview of the physiological and biochemical properties of CO and its therapeutic potential.

Heme Oxygenase-1: An Anti-Inflammatory Effector in Cardiovascular, Lung, and Related Metabolic Disorders.

The heme oxygenase (HO) enzyme system catabolizes heme to carbon monoxide (CO), ferrous iron, and biliverdin-IXα (BV), which is reduced to bilirubin-IXα (BR) by biliverdin reductase (BVR). HO activity is represented by two distinct isozymes, the inducible form, HO-1, and a constitutive form, HO-2, encoded by distinct genes (HMOX1, HMOX2, respectively). HO-1 responds to transcriptional activation in response to a wide variety of chemical and physical stimuli, including its natural substrate heme, oxidants, and phytochemical antioxidants. The expression of HO-1 is regulated by NF-E2-related factor-2 and counter-regulated by Bach-1, in a heme-sensitive manner. Additionally, HMOX1 promoter polymorphisms have been associated with human disease. The induction of HO-1 can confer protection in inflammatory conditions through removal of heme, a pro-oxidant and potential catalyst of lipid peroxidation, whereas iron released from HO activity may trigger ferritin synthesis or ferroptosis. The production of heme-derived reaction products (i.e., BV, BR) may contribute to HO-dependent cytoprotection via antioxidant and immunomodulatory effects. Additionally, BVR and BR have newly recognized roles in lipid regulation. CO may alter mitochondrial function leading to modulation of downstream signaling pathways that culminate in anti-apoptotic, anti-inflammatory, anti-proliferative and immunomodulatory effects. This review will present evidence for beneficial effects of HO-1 and its reaction products in human diseases, including cardiovascular disease (CVD), metabolic conditions, including diabetes and obesity, as well as acute and chronic diseases of the liver, kidney, or lung. Strategies targeting the HO-1 pathway, including genetic or chemical modulation of HO-1 expression, or application of BR, CO gas, or CO donor compounds show therapeutic potential in inflammatory conditions, including organ ischemia/reperfusion injury. Evidence from human studies indicate that HO-1 expression may represent a biomarker of oxidative stress in various clinical conditions, while increases in serum BR levels have been correlated inversely to risk of CVD and metabolic disease. Ongoing human clinical trials investigate the potential of CO as a therapeutic in human disease.

DAF-2c signaling promotes taste avoidance after starvation in Caenorhabditis elegans by controlling distinct phospholipase C isozymes

Previously, we reported that DAF-2c, an axonal insulin receptor isoform in Caenorhabditis elegans, acts in the ASER gustatory neuron to regulate taste avoidance learning, a process in which worms learn to avoid salt concentrations experienced during starvation. Here, we show that secretion of INS-1, an insulin-like peptide, after starvation conditioning is sufficient to drive taste avoidance via DAF-2c signaling. Starvation conditioning enhances the salt-triggered activity of AIA neurons, the main sites of INS-1 release, which potentially promotes feedback signaling to ASER to maintain DAF-2c activity during taste avoidance. Genetic studies suggest that DAF-2c–Akt signaling promotes high-salt avoidance via a decrease in PLCβ activity. On the other hand, the DAF-2c pathway promotes low-salt avoidance via PLCε and putative Akt phosphorylation sites on PLCε are essential for taste avoidance. Our findings imply that animals disperse from the location at which they experience starvation by controlling distinct PLC isozymes via DAF-2c.

Evolutionary plasticity in the allosteric regulator-binding site of pyruvate kinase isoform PykA from Pseudomonas aeruginosa

Unlike many other well-characterized bacteria, the opportunistic human pathogen Pseudomonas aeruginosa relies exclusively on the Entner-Doudoroff pathway (EDP) for glycolysis. Pyruvate kinase (PK) is the main "pacemaker" of the EDP, and its activity is also relevant for P. aeruginosa virulence. Two distinct isozymes of bacterial PK have been recognized, PykA and PykF. Here, using growth and expression analyses of relevant PK mutants, we show that PykA is the dominant isoform in P. aeruginosa Enzyme kinetics assays revealed that PykA displays potent K-type allosteric activation by glucose 6-phosphate and by intermediates from the pentose phosphate pathway. Unexpectedly, the X-ray structure of PykA at 2.4 Å resolution revealed that glucose 6-phosphate binds in a pocket that is distinct from the binding site reported for this metabolite in the PK from Mycobacterium tuberculosis (the only other available bacterial PK structure containing bound glucose 6-phosphate). We propose a mechanism by which glucose 6-phosphate binding at the allosteric site communicates with the PykA active site. Taken together, our findings indicate remarkable evolutionary plasticity in the mechanism(s) by which PK senses and responds to allosteric signals.

Abstract 3043: Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia

Abstract Myristoylation is required for biological activity of >200 intracellular proteins. N-myristoyltransferases (NMTs) transfer the fatty acid myristate to N-terminal glycine residue; there are two isoforms, NMT1 and 2. In acute myeloid leukemia (AML), upregulation of Lyn and Src, important myristoylated proteins, contribute to cell survival and proliferation. The specific roles of NMT1 and NMT2 are unknown in this context. The relationships among NMT1/NMT2 expression and acute AML patient outcomes were studied using RNA-sequencing and microarray cohorts with over 350 patients. We found that high NMT1 and low NMT2 expression were associated with reduced overall and event-free survival in adult AML, which was independent of other prognostic markers on multivariate analysis. High NMT1, but not NMT2, expression was associated with proliferative gene sets in AML cell lines, indicating potential for distinct isozyme substrates. Given these results, we examined NMT1 and NMT2 levels in AML cell lines and AML patient blast cells using Western blotting and flow cytometry. We determined that NMT2 expression varied greatly among patients, but was markedly reduced in most cases, while NMT1 expression was relatively preserved. A potent small molecule NMT inhibitor, PCLX-001, preferentially induced apoptosis and reduced viability in NMT2-deficient AML cell lines cultured in vitro compared with normal lymphocytes and peripheral blood mononuclear cells. PCLX-001 also killed freshly isolated human AML blasts ex vivo with an IC50 of ~170nM regardless of their mutational background. In a murine AML xenograft model, subcutaneously delivered PCLX-001 monotherapy demonstrated dose-dependent anticancer activity and produced complete remissions after five daily 50 mg/kg doses. NMT expression provides independent prognostic information to refine existing clinical stratification, and NMT inhibition is a promising novel therapeutic strategy for AML. Citation Format: John R. Mackey, Aishwarya Iyer, Megan C. Yap, Zoulika Zak, Krista Vincent, Erwan Beauchamp, Lynne M. Postovit, Joseph Brandwein, Luc G. Berthiaume. Examination of NMT1 and NMT2 as independent prognostic markers and novel drug targets in adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3043.

Longevity in vivo of primary cell wall cellulose synthases.

Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

Abstract 862: Atypical protein kinase c inhibitors can repress epithelial to mesenchymal transition (type III) in malignant melanoma

Abstract Melanoma is a type of cancer occurs in melanocytes. Approximately 90% of melanoma occurs in skin (cutaneous melanoma) but can rarely arise from the mucosal surfaces or areas which neural cells migrate. Examples are eye, intestine and mouth [Eur. J. Cancer, 69: 39-42 (2016)]. 76,380 of new cases and 10,130 number of deaths are expected in 2016 in the USA due to melanoma [http://seer.cancer.gov/statfacts/html/melan.html (11/05/2016)]. Atypical PKCs contains two structurally and functionally distinct isozymes in human which are PKC-ι (iota) and PKC-ζ (zeta). They are believed to be involved in cell cycle progression, tumorigenesis, cell survival and cell migration. We believe that atypical PKCs play an important role in cell motility of melanoma by involving the signaling pathways which induces EMT-type III (Epithelial to Mesenchymal Transition). In normal melanocytes, PKC-ζ was found in low levels and PKC-ι was not detected. But both proteins are detected in very high levels in malignant melanoma [Melenoma Res. 12:201-209 (2002)]. In the current study, we have investigated the effects of novel atypical PKC inhibitors [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) which is specific to PKC-ι and 8-hydroxy-1, 3, 6-naphthalenetrisulfonic acid (Compound-50) which is specific to PKC-ζ on the cell proliferation, apoptosis and cell migration of two malignant melanoma cell lines (SK-MEL-2 and MeWo) compared to a normal melanocyte cell line (PCS-200-013). We showed that both inhibitors can decrease the levels of total and phosphorylated levels of PKC-ζ and PKC-ι. Furthermore, both inhibitors increased the levels of E-cadherin and decreased the levels of Vimentin which is a mesenchymal marker associated with EMT. Treatments with inhibitors altered the levels of CD44, a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, migration and tumor cell homing during metastasis. These results suggest that both PKC-ι and PKC-ζ are involved in signaling pathways which upregulate EMT and which can be effectively suppress using ICA-1 and Compound-50. Furthermore we established that treatment with ICA-1/Compound-50 induced apoptosis as shown by increasing Caspase-3 levels and decreasing Bcl-2 levels. Citation Format: Wishrawana Sarathi Ratnayake, Mildred Acevedo-Duncan. Atypical protein kinase c inhibitors can repress epithelial to mesenchymal transition (type III) in malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 862. doi:10.1158/1538-7445.AM2017-862

Specific peroxidases differentiate Brachypodium distachyon accessions and are associated with drought tolerance traits.

Brachypodium distachyon (Brachypodium) is a model system for studying cereal, bioenergy, forage and turf grasses. The genetic and evolutionary basis of the adaptation of this wild grass species to drought stress is largely unknown. Peroxidase (POD) may play a role in plant drought tolerance, but whether the allelic variations of genes encoding the specific POD isoenzymes are associated with plant response to drought stress is not well understood. The objectives of this study were to examine natural variation of POD isoenzyme patterns, to identify nucleotide diversity of POD genes and to relate the allelic variation of genes to drought tolerance traits of diverse Brachypodium accessions. Whole-plant drought tolerance and POD activity were examined in contrasting ecotypes. Non-denaturing PAGE and liquid chromatography-mass spectrometry were performed to detect distinct isozymes of POD in 34 accessions. Single nucleotide polymorphisms (SNPs) were identified by comparing DNA sequences of these accessions. Associations of POD genes encoding specific POD isoenzymes with drought tolerance traits were analysed using TASSEL software. Variations of POD isoenzymes were found among accessions with contrasting drought tolerance, while the most tolerant and susceptible accessions each had their own unique POD isoenzyme band. Eight POD genes were identified and a total of 90 SNPs were found among these genes across 34 accessions. After controlling population structure, significant associations of Bradi3g41340.1 and Bradi1g26870.1 with leaf water content or leaf wilting were identified. Brachypodium ecotypes have distinct specific POD isozymes. This may contribute to natural variations of drought tolerance of this species. The role of specific POD genes in differentiating Brachypodium accessions with contrasting drought tolerance could be associated with the general fitness of Brachypodium during evolution.

Inhibition of branched chain amino acid aminotransferase 1 suppresses the severity of collagen-induced arthritis in mice (THER5P.905)

Abstract BCAT is the enzyme responsible for the reverse transamination of branched chain amino acids to corresponding α-keto acids. It exists as two distinct isozymes with mutually exclusive tissue distribution: a mitochondrial one (BCAT2) and a cytosolic one (BCAT1). The present study reports on the effect of BCAT1 inhibition on CIA, a murine model of rheumatoid arthritis (RA). Oral administration of ERG240, a small molecular weight BCAT1 inhibitor, alleviated the severity of the clinical development of CIA and exerted a strong protective effect on the integrity of the joints of affected animals. When the drug was given at 1000 mg/kg 5x per week for 4 weeks the clinical score and paw thickness was reduced by 40%. Immunohistochemical (IHC) analysis of preserved tissues revealed that ERG240 treatment reduced inflammation by ~50%, pannus formation by ~90%, cartilage damage by ~75%, and bone erosion by ~90%. Further IHC analysis indicated that the mechanism of action of ERG240 involved a reduction in the number of macrophages and osteoclasts present in the joints and CD45R+ cells in the bone marrow of the animals. Analysis of terminal sera, collected from treated and control animals, suggested that ERG240 administration also suppresses the levels of secreted cytokines such as TNF-α and RANKL. In vitro, ERG240 inhibited serum-driven migration of bone marrow derived macrophages. Collectively, the data indicate that BCAT1 constitutes a new druggable target for the treatment of RA.

Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues.

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the “amidated” and “deamidated” routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

Protein kinase C in the wood frog, Rana sylvatica: reassessing the tissue-specific regulation of PKC isozymes during freezing.

The wood frog, Rana sylvatica, survives whole-body freezing and thawing each winter. The extensive adaptations required at the biochemical level are facilitated by alterations to signaling pathways, including the insulin/Akt and AMPK pathways. Past studies investigating changing tissue-specific patterns of the second messenger IP3 in adapted frogs have suggested important roles for protein kinase C (PKC) in response to stress. In addition to their dependence on second messengers, phosphorylation of three PKC sites by upstream kinases (most notably PDK1) is needed for full PKC activation, according to widely-accepted models. The present study uses phospho-specific immunoblotting to investigate phosphorylation states of PKC—as they relate to distinct tissues, PKC isozymes, and phosphorylation sites—in control and frozen frogs. In contrast to past studies where second messengers of PKC increased during the freezing process, phosphorylation of PKC tended to generally decline in most tissues of frozen frogs. All PKC isozymes and specific phosphorylation sites detected by immunoblotting decreased in phosphorylation levels in hind leg skeletal muscle and hearts of frozen frogs. Most PKC isozymes and specific phosphorylation sites detected in livers and kidneys also declined; the only exceptions were the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641) antibody, which remained unchanged from control to frozen frogs. Changes in brains of frozen frogs were unique; no decreases were observed in the phosphorylation levels of any of the PKC isozymes and/or specific phosphorylation sites detected by immunoblotting. Rather, increases were observed for the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641), phospho-PKCδ (Thr505), and phospho-PKCθ (Thr538) antibodies; all other isozymes/phosphorylation sites detected in brain remained unchanged from control to frozen frogs. The results of this study indicate a potential important role for PKC in cerebral protection during wood frog freezing. Our findings also call for a reassessment of the previously-inferred importance of PKC in other tissues, particularly in liver; a more thorough investigation is required to determine whether PKC activity in this physiological situation is indeed dependent on phosphorylation, or whether it deviates from the generally-accepted model and can be “overridden” by exceedingly high levels of second messengers, as has been demonstrated with certain PKC isozymes (e.g., PKCδ).

Human Nitrilase-like Protein Does Not Catalyze the Hydrolysis of Vildagliptin

Nitrilase, which is found in plants and many types of bacteria, is known as the enzyme that catalyzes hydrolysis of a wide variety of nitrile compounds. While human nitrilase-like protein (NIT), which is a member of the nitrilase superfamily, has two distinct isozymes, NIT1 and NIT2, their function has not been well understood. In this study, we investigated whether human NIT1 and NIT2 are involved in the hydrolysis of drugs using vildagliptin as a substrate. We performed Western blot analysis using human liver samples to examine protein expression of human NIT in the liver, finding that human NIT1 and NIT2 were highly expressed in the liver cytosol. We established stable single expression systems of human NIT1 and NIT2 in HEK293 cells to clarify the contribution of human NIT to hydrolysis of vildagliptin. Although the formation of a carboxylic acid metabolite of vildagliptin (M20.7) was observed in human liver samples, M20.7 was not formed by incubating vildagliptin with HEK293 cells expressing human NIT1 or NIT2. This suggests that human NIT1 or NIT2 is not involved in the metabolism of vildagliptin. Further investigation using other drugs is needed to clarify the contribution of human NIT to drug metabolism.

Evidence for expression of 11β-hydroxysteroid dehydrogenase type3 (HSD11B3/HSD11B1L) in neonatal pig testis

11β-hydroxysteroid dehydrogenase (HSD11B) catalyzes the interconversion between active and inactive glucocorticoid, and is known to exist as two distinct isozymes: HSD11B1 and HSD11B2. A third HSD11B isozyme, HSD11B1L (SCDR10b), has recently been identified. Human HSD11B1L, which was characterized as a unidirectional NADP(+)-dependent cortisol dehydrogenase, appears to be specifically expressed in the brain. We previously reported that HSD11B1 and abundant HSD11B2 isozymes are expressed in neonatal pig testis and the Km for cortisol of NADP(+)-dependent dehydrogenase activity of testicular microsomes obviously differs from the same activity catalyzed by HSD11B1 from pig liver microsomes. Therefore, we hypothesized that the neonatal pig testis also expresses the third type of HSD11B isozyme, and we herein examined further evidence regarding the expression of HSD11B1L. (1) The inhibitory effects of gossypol and glycyrrhetinic acid on pig testicular microsomal NADP(+)-dependent cortisol dehydrogenase activity was clearly different from that of pig liver microsomes. (2) A highly conserved human HSD11B1L sequence was observed by RT-PCR in a pig testicular cDNA library. (3) mRNA, which contains the amplified sequence, was evaluated by real-time PCR and was most strongly expressed in pig brain, and at almost the same levels in the kidney as in the testis, but at lower levels in the liver. Based on these results, neonatal pig testis appears to express glycyrrhetinic acid-resistant HSD11B1L as a third HSD11B isozyme, and it may play a physiologically important role in cooperation with the abundantly expressed HSD11B2 isozyme in order to prevent Leydig cell apoptosis or GC-mediated suppression of testosterone production induced by high concentrations of activated GC in neonatal pig testis.

Differential contribution of lipoxygenase isozymes to nigrostriatal vulnerability

The 5- and 12/15-lipoxygenase (LOX) isozymes have been implicated to contribute to disease development in CNS disorders such as Alzheimer’s disease. These LOX isozymes are distinct in function, with differential effects on neuroinflammation, and the impact of the distinct isozymes in the pathogenesis of Parkinson’s disease has not as yet been evaluated. To determine whether the isozymes contribute differently to nigrostriatal vulnerability, the effects of 5- and 12/15-LOX deficiency on dopaminergic tone under naïve and toxicant-challenged conditions were tested. In naïve mice deficient in 5-LOX expression, a modest but significant reduction (18.0% reduction vs. wildtype (WT)) in striatal dopamine (DA) was detected (n=6–8 per genotype). A concomitant decline in striatal tyrosine hydroxylase (TH) enzyme was also revealed in null 5-LOX vs. WT mice (26.2%); however, no changes in levels of DA or TH immunoreactivity were observed in null 12/15-LOX vs. WT mice. When challenged with the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), WT mice showed a marked reduction in DA (31.9%) and robust astrocytic and microglial activation as compared to saline-treated animals. In contrast, null 5-LOX littermates demonstrated no significant striatal DA depletion or astrogliosis (as noted by Western blot analyses for glial acidic fibrillary protein (GFAP) immunoreactivity). In naïve null 12/15-LOX mice, no significant change in striatal DA values was observed compared to WT, and following MPTP treatment, the transgenics revealed striatal DA reduction similar to the challenged WT mice. Taken together, these data provide the first evidence that: (i) LOX isozymes are involved in the maintenance of normal dopaminergic function in the striatum and (ii) the 5- and 12/15-LOX isozymes contribute differentially to striatal vulnerability in response to neurotoxicant challenge.

A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV)

A new continuous cell line, NTU-SE, was established from the pupal tissues of an economically important pest, the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). This cell line contains four major morphologic types: round, polymorphic, spindle-shaped, and comma-shaped cells. The population doubling time of this new line in TNM-FH medium supplemented with 8% fetal bovine serum (FBS) at 28°C is 35.5h. The chromosomal spread from NTU-SE cells is typical to the chromosomal morphology of lepidopteran cell lines. Confidently, NTU-SE cell line is a new cell line that exhibits distinct isozyme patterns of esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) from those of the other insect cell lines. In addition, the DNA sequence of the nuclear ribosomal internal transcribed spacer (ITS) region of NTU-SE cells is above 96% identical to that sequence of S. exigua larvae, as compared to only 66% identical to that of S. litura larvae. The NTU-SE cell line is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). Therefore, a highly virulent SeMNPV strain, SeMNPV-1, had been successfully isolated and propagated in NTU-SE cells. We conclude that the NTU-SE cell line will be a useful tool for the selection and mass production of highly virulent SeMNPV strains for the S. exigua biocontrol and the baculovirus based recombinant protein expression systems.

Expression of aldehyde dehydrogenase gene increases hydrogen production from low concentration of acetate by Rhodobacter sphaeroides

Rhodobacter sphaeroides RV (RV) is a hydrogen-producing bacterium exhibiting the highest yield of hydrogen production from organic acids such as lactate and acetate, which are the byproducts of hydrogen fermentation by hydrogen-producing anaerobic bacteria. Co-fermentation of the RV strain with anaerobic bacteria is an efficient method of hydrogen production. However, less than 21 mM acetate is produced by the anaerobic bacteria, which is too low for efficient hydrogen production by the RV strain; it requires approximately 75 mM acetate. In this study, 2 distinct isozymes of aldehyde dehydrogenase from Rhodospirillum rubrum were separately overexpressed in the RV strain. The recombinant RV strains that were designated as RVAD1 and RVAD2, exhibited 13-fold higher ALDH activities than the wild-type RV strain. Hydrogen yields of both of the recombinant strains were 1.4-fold higher than that of the RV strain in 21 mM acetate. In 43 mM acetate, the RVAD1 strain showed higher yield, though the RVAD2 strain showed lower yield as compared to that of the RV strain. In 64 mM acetate and all concentrations of lactate tested (21, 43 and 64 mM), the yields of the recombinant strains were lower than those of the RV strain. The intact (empty) expression plasmid increased the ALDH activity and had little effect on the hydrogen production in acetate, however, it decreased the production in lactate. At the beginning of the fermentation process, when very little hydrogen had been produced, the recombinant strains expressing the ALDH gene consumed smaller amounts of acetate compared to the wild-type strain. We have discussed the effects of ALDH on hydrogen production in this report.

Xylanase Isozymes from the Newly Isolated Bacillus sp. CKBx1D and Optimization of Its Deinking Potentiality

Recycling of civic paper waste by enzyme-based technology is nowadays a point of much concern for pollution-less green environment. In this study, the deinking effectiveness of purified xylanase from a newly isolated bacterium was evaluated for recycling of laser jet paper waste. A potent xylanases-producing bacterium from the microbial consortia of termite gut was isolated, which was further identified on the basis of 16S rRNA sequence as Bacillus sp. CKBx1D. In submerged fermentation condition, the isolate produced the highest level of xylanase (480U/ml) at 36h of growth. The extracellular xylanase system comprises of three distinct isozymes (est. Mw 35.28, 28.63, 18.94kDa). The deinking of laser printed paper waste was performed using the purified enzyme mixture. Whole operational parameters were optimized using the Response Surface Methodology; it was found that at pH 6.8 with 47.2h of continuous shaking at constant temperature of 35°C, enzymes showed best deinking activity. After enzyme treatment, the physical properties of the pulp like brightness and ERIC (effective residual ink content) values were enhanced, whereas the pulp opacity was more reduced than the control treatment. Hence, the bacterial isolate and its xylanolytic enzyme system could efficiently be used in recycling paper waste as deinking agent.

PDE1 isozymes, key regulators of pathological vascular remodeling

Pathological vascular remodeling is a hallmark of most vascular disorders such as atherosclerosis, postangioplasty restenosis, allograft vasculopathy, and pulmonary hypertension. Pathological vascular remodeling is a multi-cell-dependent process leading to detrimental changes of vessel structure and eventual vessel occlusion. Cyclic nucleotide signaling regulates a variety of vascular functions ranging from cell contractility to cell growth. Cyclic nucleotide phosphodiesterases (PDEs), a large family of structurally and functionally distinct isozymes, regulate cyclic nucleotide levels and compartmentalization through catalyzing their degradation reaction. Increasing evidence has suggested that one of the important mechanisms for specific cyclic nucleotide regulation is exerted through selective activation or inhibition of distinct PDE isozymes. This review summarizes the work done to characterize the role and therapeutic potential of PDE1 isozymes in pathological vascular remodeling.