Dehydrogenase Isozymes Research Articles

Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme.

Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development. This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility. Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function. Homology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose-response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell. Our studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD(+)-bound human GAPDHS, facilitating the identification of unique structural features of this sperm isozyme. In dose-response assays T0501_7749 inhibited human GAPDHS with an IC50 of 1.2 μM compared with an IC50 of 38.5 μM for the somatic isozyme. This compound caused significant reductions in mouse sperm lactate production (P= 0.017 for 100 μM T0501_7749 versus control) and in the percentage of motile mouse and human sperm (P values from <0.05 to <0.0001, depending on incubation conditions). The chemical properties of T0501_7749, including limited solubility and nonspecific protein binding, are not optimal for drug development. This study provides proof-of-principle evidence that GAPDHS can be selectively inhibited, causing significant reductions in sperm glycolysis and motility. These results highlight the utility of structure-based drug design and support further exploration of GAPDHS, and perhaps other sperm-specific isozymes in the glycolytic pathway, as contraceptive targets. None. Coordinates and data files for three GAPDHS crystal structures were deposited in the RCSB Protein Data Bank (http://www.rcsb.org). This work was supported by grants from the National Institutes of Health (NIH), USA, including U01 HD060481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and TW/HD00627 from the NIH Fogarty International Center. Additional support was provided by subproject CIG-05-109 from CICCR, a program of CONRAD, Eastern Virginia Medical School, USA. There are no conflicts of interest.

Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C₄ of the Plateau Pika (Ochotona curzoniae).

Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4.

BET protein inhibition mitigates acute myocardial infarction damage in rats via the TLR4/TRAF6/NF-κB pathway.

Acute myocardial infarction (AMI) is among the most serious cardiovascular diseases and is a leading cause of mortality in developed countries. Previous studies have indicated the central role played by the bromodomain (BRD) proteins, which belong to the BRD and extra-terminal (BET) family, in gene control during heart failure pathogenesis. In addition, BET inhibition has been shown to suppress cardiomyocyte hypertrophy. However, the role of BET proteins in myocardial infarction remains unclear. The present study aimed to investigate whether BETs inhibition mitigates AMI, and explore the molecular mechanism underlying this effect. A rat model of acute myocardial infarction was established, and rats were divided into the sham, AMI and AMI + JQ1 groups. JQ1, a well-known selective BRD inhibitor, was used to suppress BET domain family activity. The mRNA and protein expression levels of BRD2, BRD3 and BRD4 were evaluated using quantitative polymerase chain reaction and western blot analysis, respectively. In addition, the expression levels of markers of cardiac damage were determined using commercial kits. The results indicated that BRD2 and BRD4 mRNA and protein expression levels were significantly increased in the AMI group compared with those in the sham group. In addition, BET inhibition decreased AMI damage in vivo by reversing cardiac function injury, decreasing serum lactate dehydrogenase and creatine kinase-MB isozyme activity, in addition to decreasing the expression levels of high-sensitivity C-reactive protein and interleukin-6. Furthermore, the results suggested that Toll-like receptor 4 (TLR4) signaling was activated by the increased expression of TLR4, TNF receptor-associated factor 6 (TRAF6) and nuclear factor (NF)-κB during AMI. However, JQ1 treatment suppressed TLR4 signaling activation. In conclusion, the present results demonstrated that the inhibition of BET family proteins suppresses AMI, and that this effect was partially mediated by the inhibition of TLR4/TRAF6/NF-κB signaling.

Topically applied metal chelator reduces thermal injury progression in a rat model of brass comb burn

Oxidative stress may be involved in the cellular damage and tissue destruction as burn wounds continues to progress after abatement of the initial insult. Since iron and calcium ions play key roles in oxidative stress, this study tested whether topical application of Livionex formulation (LF) lotion, that contains disodium EDTA as a metal chelator and methyl sulfonyl methane (MSM) as a permeability enhancer, would prevent or reduce burns. MethodsWe used an established brass comb burn model with some modifications. Topical application of LF lotion was started 5min post-burn, and repeated every 8h for 3 consecutive days. Rats were euthanized and skin harvested for histochemistry and immunohistochemistry. Formation of protein adducts of 4-hydroxynonenal (HNE), malonadialdehyde (MDA) and acrolein (ACR) and expression of aldehyde dehydrogenase (ALDH) isozymes, ALDH1 and ALDH2 were assessed. ResultsLF lotion-treated burn sites and interspaces showed mild morphological improvement compared to untreated burn sites. Furthermore, the lotion significantly decreased the immunostaining of lipid aldehyde–protein adducts including protein -HNE, -MDA and -ACR adducts, and restored the expression of aldehyde dehydrogenase isozymes in the unburned interspaces. ConclusionThis data, for the first time, demonstrates that a topically applied EDTA-containing lotion protects burns progression with a concomitant decrease in the accumulation of reactive lipid aldehydes and protection of aldehyde dehydrogenase isozymes. Present studies are suggestive of therapeutic intervention of burns by this novel lotion.

Evidence of High Gene Flow Between Samples of Horseweed (Conyza canadensis) and Hairy Fleabane (Conyza bonariensis) as Revealed by Isozyme Polymorphisms

Native polyacrylamide gel electrophoresis was used to identify polymorphisms in α- and β-esterases loci and electrophoresis in starch gel to identify polymorphism in malate dehydrogenase (MDH; EC 1.1.1.37) and acid phosphatase (ACP; EC 3.1.3.2) isozymes loci in leaf tissues from samples of horseweed and hairy fleabane populations to determine genetic diversity and population structure. Similar or differential genetic divergence between the two species may guide specific use of herbicides. For samples of plants with high genetic similarity it is possible to adopt similar mechanisms and processes for their control. The proportion of polymorphic loci was 57.14, 50.0, and 53.6%, in samples of horseweed and hairy fleabane, for EST, MDH, and ACP isozymes, respectively. A comparison of the diversity parameters in the two species showed that the number of alleles is similar in the horseweed and hairy fleabane plants. The estimated heterozygosity in horseweed and hairy fleabane was also very close. A relatively low level of population differentiation was detected between horseweed and hairy fleabane (FST= 0.0199), which suggests a substantial genetic exchange among the two species. Accordingly, estimate of gene flow was high (Nm = 12.3172) for the alleles of the lociEst,Mdh, andAcp. The Nei's identity (I) values also was high (I = 0.9561) indicating very high similarity between the twoConyzaspecies. AMOVA showed higher genetic variation within (95%) than among (5%) the two samples. The low genetic structure and high value of genetic identity was an important indication that alleles are exchanged between horseweed and hairy fleabane populations, and provides additional evidence of occurrence of outcrossing between populations or dispersion of samples of one for other site.

More than mitochondrial biogenesis: alternative roles of PGC-1α in exercise adaptation.

More than mitochondrial biogenesis: alternative roles of PGC-1α in exercise adaptation.

Long-term expression of metabolism-associated genes in the rat hippocampus following recurrent neonatal seizures and its regulation by melatonin

Despite the effective use of antiepileptic drugs (AEDs) for epilepsy, therapeutic failure occurs in 30% of patients. Novel approaches are targeting the inhibition of epileptogenesis. N-acetyl-5-methoxytryptamine (melatonin) is an indoleamine produced mainly by the pineal gland, and has been observed to exhibit antiepileptic and neuroprotective effects in experimental and clinical investigations. In the present study, the underlying protective mechanism of melatonin on neonatal seizure-induced long-term excitotoxicity was examined in the hippocampus of rats, predominantly on the metabolism-associated genes. Sprague Dawley rats (6-day-old; P6) were randomly divided into four groups, the control (Cont), melatonin-treated control (Mel), recurrent neonatal seizure (RS) and treatment with melatonin and RS combined (Mel+RS). At P35, mossy fiber sprouting and changes in gene expression in hippocampus were assessed using Timm staining, reverse transcription-quantitative polymerase chain reaction and use of the 2(-ΔCT) methods, respectively. The aberrant mossy fiber sprouting in the supra granular region of the dentate gyrus and CA3 subfield of the hippocampus was suppressed by pretreatment with melatonin. In addition, among the nineteen genes identified, four energy metabolism-associated genes (Kcnj11, leptin receptor, dopamine receptor D2 and melanocortin 4 receptor), four lipid metabolism-associated genes (apolipoprotein A-I, opioid receptor κ 1, pyruvate dehydrogenase kinase, isozyme 4 and cytochrome P450, family 46, subfamily a, polypeptide 1) and zinc transporter 1 (ZnT1), sphingomyelinase (nSMase) and Cathepsin-E, were markedly downregulated by melatonin treatment in the Mel group or in the developmental seizure RS and Mel + RS groups, compared with that in the Cont group. Furthermore, the melatonin-pretreated seizure rats (Mel + RS) exhibited a significantly upregulated expression of calcium/calmodulin-dependent protein kinase II α (CaMKIIα), acetyl-Coenzyme A acetyltransferase 1 (ACAT1), ZnT-1, metallothionein 1 (MT-1), nSMase and Cathepsin-E, compared with the RS rats. Thus, the present study investigated changes in the expression of metabolic genes in the hippocampus following pretreatment with melatonin. Fluorthyl-induced decreases in the expression levels of ACAT1/nSMase/Cathepsin-E, ZnT-1/MT-1 and CaMKIIα in the hippocampus, and the reversal by melatonin may be associated with a decrease in neonatal seizure-induced aberrant mossy fiber sprouting, which requires further investigation.

Sex-dependent effects of developmental arsenic exposure on methylation capacity and methylation regulation of the glucocorticoid receptor system in the embryonic mouse brain

Previously we have shown that prenatal moderate arsenic exposure (50 ppb) disrupts glucocorticoid receptor (GR) programming and that these changes continue into adolescence in males. However, it was not clear what the molecular mechanisms were promoting these GR programming changes or if these changes occurred in arsenic-exposed females. In the present studies, we assessed the effects of arsenic on protein and mRNA of the glucocorticoid receptor (GR) and 11β-hydroxysteroid dehydrogenase (Hsd) isozymes and compared the levels of methylation within the promoters of the Nr3c1 and Hsd11b1 genes in female fetal brain at embryonic days (E) 14 and 18. Prenatal arsenate exposure produced sex specific effects on the glucocorticoid system. Compared to males, females were resistant to arsenic induced changes in GR, 11β-Hsd-1 and 11β-Hsd-2 protein levels despite observed elevations in Nr3c1 and Hsd11b2 mRNA. This sex-specific effect was not due to differences in the methylation of the GR promoter as methylation of the Nr3c1 gene was either unchanged (region containing the egr-1 binding site) or similarly reduced (region containing the SP-1 transcription factor binding site) in both males and females exposed to arsenic. Arsenic did produce sex and age-specific changes in the methylation of Hsd11b1 gene, producing increased methylation in females at E14 and decreased methylation at E18.These changes were not attributed to changes in DNMT levels. Since arsenate metabolism could interfere with the generation of methyl donor groups, we assessed glutathione (GSH), S-adenosylmethionine (SAM) and As 3 methyltransferase (As3MT). Exposed males and females had similar levels of As3MT and SAM; however, females had higher levels of GSH/GSSH. It is possible that this greater anti-oxidative capacity within the females provides protection against low to moderate arsenate. Our data suggest that the GR signaling system in female offspring was not as affected by prenatal arsenic and predicts that female arsenic-exposed mice should have normal GR feedback regulation.

Meloidogyne lopezi n. sp. (Nematoda: Meloidogynidae), a new root-knot nematode associated with coffee (Coffea arabica L.) in Costa Rica, its diagnosis and phylogenetic relationship with other coffee-parasitising Meloidogyne species

Coffee (Coffea arabica L. cv. Catuai) seedlings with abundant small root galls caused by an unknown root-knot nematode were found in southern Costa Rica. Morphology, esterase and malate dehydrogenase isozyme phenotypes and DNA markers differentiated this nematode from known Meloidogyne spp. A new species, M. lopezi n. sp., with common name Costa Rican root-knot nematode, is suggested. Meloidogyne lopezi n. sp. is distinguished from other coffee-associated Meloidogyne spp. by size of female lips and stylet, male body length and stylet and second-stage juvenile body and tail morphology. The region of the mitochondrial genome between COII and 16S rRNA showed a unique amplicon size of 1370 bp, and digestions with restriction enzymes HinfI, AluI, DraI and DraIII revealed characteristic PCR-RFLP patterns that differed from the tropical root-knot nematode species M. arabicida, M. incognita, M. izalcoensis, M. javanica and M. paranaensis. Characterisation of the protein-coding map-1 gene and phylogenetic analyses suggested that M. lopezi n. sp. might reproduce by mitotic parthenogenesis. Phylogenies estimated using Bayesian analyses based on the region between the COII and 16S rRNA mitochondrial genes, as well as the 18S and 28S ribosomal nuclear genes, indicated that M. lopezi n. sp. is closely related to other tropical Meloidogyne spp. that infect coffee, especially M. arabicida, M. izalcoensis and M. paranaensis from Central and South America. Isozyme analyses and PCR-RFLP of the COII-16S rRNA mitochondrial gene region enable a clear diagnostic differentiation between these species.

Geographic and climatic differentiation of electrophoretic forms of esterase, glutamate dehydrogenase and peroxidase in Scots pine tissues

The enzymatic systems have been investigated in individual trees derived from 17 distant localities in Europe and Asia. Esterase and glutamate dehydrogenase polymorphism was determined in extracts from mixture of 50 macrogametophytes, iperoxidase polymorphism was investigated in needle extracts from 3-month-old seedlings. While testing by disc electrophoresis needle extracts of Scots pine deriving from specimens of different geographic ranges it was found that those from the South displayed mare isoperoxidases than those from the North. Most pronounced differences in number and relative activity of esterase and glutamate dehydrogenase isozymes were found in the specimens originating from isolated stands. It is suggested that hight temperatures and dry summer periods could influence the number and activity of the isozymes.

On the species status of the root-knot nematode Meloidogyne ulmi Palmisano &amp; Ambrogioni, 2000 (Nematoda, Meloidogynidae)

The root-knot nematode Meloidogyne ulmi is synonymised with Meloidogyne mali based on morphological and morphometric similarities, common hosts, as well as biochemical similarities at both protein and DNA levels. M. mali was first described in Japan on Malus prunifolia Borkh.; and M. ulmi in Italy on Ulmus chenmoui W.C. Cheng. Morphological and morphometric studies of their holo- and paratypes revealed important similarities in the major characters as well as some general variability in a few others. Host test also showed that besides the two species being able to parasitize the type hosts of the other, they share some other common hosts. Our study of the esterase and malate dehydrogenase isozyme phenotypes of some M. ulmi populations gave a perfectly comparable result to that already known for M. mali. Finally, phylogenetic studies of their SSU and LSU rDNA sequence data revealed that the two are not distinguishable at DNA level. All these put together, leave strong evidences to support the fact that M. ulmi is not a valid species, but a junior synonym of M. mali. Brief discussion on the biology and life cycle of M. mali is given. An overview of all known hosts and the possible distribution of M. mali in Europe are also presented.

Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification

A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

Inhibition of human alcohol and aldehyde dehydrogenases by acetaminophen: Assessment of the effects on first-pass metabolism of ethanol

Acetaminophen is one of the most widely used over-the-counter analgesic, antipyretic medications. Use of acetaminophen and alcohol are commonly associated. Previous studies showed that acetaminophen might affect bioavailability of ethanol by inhibiting gastric alcohol dehydrogenase (ADH). However, potential inhibitions by acetaminophen of first-pass metabolism (FPM) of ethanol, catalyzed by the human ADH family and by relevant aldehyde dehydrogenase (ALDH) isozymes, remain undefined. ADH and ALDH both exhibit racially distinct allozymes and tissue-specific distribution of isozymes, and are principal enzymes responsible for ethanol metabolism in humans. In this study, we investigated acetaminophen inhibition of ethanol oxidation with recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and inhibition of acetaldehyde oxidation with recombinant human ALDH1A1 and ALDH2. The investigations were done at near physiological pH 7.5 and with a cytoplasmic coenzyme concentration of 0.5 mm NAD+. Acetaminophen acted as a noncompetitive inhibitor for ADH enzymes, with the slope inhibition constants (Kis) ranging from 0.90 mm (ADH2) to 20 mm (ADH1A), and the intercept inhibition constants (Kii) ranging from 1.4 mm (ADH1C allozymes) to 19 mm (ADH1A). Acetaminophen exhibited noncompetitive inhibition for ALDH2 (Kis = 3.0 mm and Kii = 2.2 mm), but competitive inhibition for ALDH1A1 (Kis = 0.96 mm). The metabolic interactions between acetaminophen and ethanol/acetaldehyde were assessed by computer simulation using inhibition equations and the determined kinetic constants. At therapeutic to subtoxic plasma levels of acetaminophen (i.e., 0.2–0.5 mm) and physiologically relevant concentrations of ethanol (10 mm) and acetaldehyde (10 μm) in target tissues, acetaminophen could inhibit ADH1C allozymes (12–26%) and ADH2 (14–28%) in the liver and small intestine, ADH4 (15–31%) in the stomach, and ALDH1A1 (16–33%) and ALDH2 (8.3–19%) in all 3 tissues. The results suggest that inhibition by acetaminophen of hepatic and gastrointestinal FPM of ethanol through ADH and ALDH pathways might become significant at higher, subtoxic levels of acetaminophen.

Can Protein Conformers Be Fractionated by Crystallization?

Molecular crystallization typically singles out a specific conformation, or a set of conformations that are identical over large parts and may show some flexibility, from a mixture of equilibrating conformations in solution. To critically evaluate the selectivity of this process, human lactate dehydrogenase isozyme 1 (LDH-1) microcrystals were separately dissolved and subsequently assayed inside capillaries with electrophoretically mediated microanalysis (EMMA) at both the ensemble and the single-molecule level. While fragments from the same crystal exhibited identical enzyme activities, different crystals, even when grown from the same drop of mother liquor, showed markedly different activities. Activities of individual molecules from a crystal were found to be essentially identical, whereas molecules obtained directly from solution showed a 4-fold variation in activity. Furthermore, after storage at 37 °C, the distribution of single-molecule LDH activities from solutions of individual crystals broadened and approached that of LDH obtained from the original solution. X-ray crystallography also showed distinct conformations for single microcrystals and confirms that crystallization properly selects even small conformational variants of proteins and that the slow equilibration to multiple stable conformations in solution is responsible for the observed single-molecule heterogeneity.

Moderate grade hyperammonemia activates lactate dehydrogenase-4 and 6-phosphofructo-2-kinase to support increased lactate turnover in the brain slices

Rapid metabolism of lactate is an important aspect of bioenergetic adaptation in the brain during non-physiological conditions. The low grade hyperammonemia (HA) is a common condition in the patients with chronic hepatic encephalopathy (HE); however, biochemistry of lactate turnover during low grade HA remains poorly defined. The present article describes profile of lactate dehydrogenase (LDH) isozymes vis-a-vis lactate level in the brain slices exposed with 0.1-0.5 mM ammonia, found to exist in the brain during chronic HE. A significant increment in LDH activity coincided with a similar increase in lactate level in the brain slices exposed with 0.5 mM ammonia. This was consistent with a selective increment of LDH-4 that synthesizes lactate from pyruvate with a concomitant decline in LDH-1 which catalyzes conversion of lactate to pyruvate; resulting into ~3-fold increase in LDH-4/LDH-1 ratio in those brain slices. The PFK2 domain of PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) regulates glycolysis to maintain the pyruvate pool for lactate synthesis. The PFK2 expression was also observed to be increased ~2-fold (P < 0.001) in 0.5 mM ammonia treated brain slices. These findings provide enzymatic regulation of increased lactate turnover in the brain exposed with moderate HA.

Cloning, Expression and Characterization of 3-Hydroxyisobutyrate Dehydrogenase from Pseudomonas denitrificans ATCC 13867

The gene encoding an NAD+-dependent, 3-hydroxyisobutyrate dehydrogenase (3HIBDH-IV) from Pseudomonas denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL 21 (DE3) and characterized to understand its physiological relevance in the degradation of 3-hydroxypropionic acid (3-HP). The deduced amino acid sequence showed high similarity to other 3-hydroxyisobutyrate dehydrogenase isozymes (3HIBDHs) of P. denitrificans ATCC 13867. A comparison of 3HIBDH-IV with its relevant enzymes along with molecular docking studies suggested that Lys171, Asn175 and Gly123 are important for its catalytic function on 3-hydroxyacids. The recombinant 3HIBDH-IV was purified to homogeneity utilizing a Ni-NTA-HP resin column in high yield. 3HIBDH-IV was very specific to (S)-3-hydroxyisobutyrate, but also catalyzed the oxidation of 3-HP to malonate semialdehyde. The specific activity and half-saturation constant (K m) for 3-HP at 30°C and pH 9.0 were determined to be 17 U/mg protein and 1.0 mM, respectively. Heavy metals, such as Ag+ and Hg2+, completely inhibited the 3HIBDH-IV activity, whereas dithiothreitol, 2-mercaptoethanol and ethylenediaminetetraacetic acid increased its activity 1.5–1.8-fold. This paper reports the characteristics of 3HIBDH-IV as well as its probable role in 3-HP degradation.

Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.

Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.

A Systematic Approach to the Diagnosis of Suspected Central Nervous System Lymphoma

Central nervous system (CNS) lymphoma can present a diagnostic challenge. Currently, there is no consensus regarding what presurgical evaluation is warranted or how to proceed when lesions are not surgically accessible. We conducted a review of the literature on CNS lymphoma diagnosis (1966 to October 2011) to determine whether a common diagnostic algorithm can be generated. We extracted data regarding the usefulness of brain and body imaging, serum and cerebrospinal fluid (CSF) studies, ophthalmologic examination, and tissue biopsy in the diagnosis of CNS lymphoma. Contrast enhancement on imaging is highly sensitive at the time of diagnosis: 98.9% in immunocompetent lymphoma and 96.1% in human immunodeficiency virus-related CNS lymphoma. The sensitivity of CSF cytology is low (2%-32%) but increases when combined with flow cytometry. Cerebrospinal fluid lactate dehydrogenase isozyme 5, β2-microglobulin, and immunoglobulin heavy chain rearrangement studies have improved sensitivity over CSF cytology (58%-85%) but have only moderate specificity (85%). New techniques of proteomics and microRNA analysis have more than 95% specificity in the diagnosis of CNS lymphoma. Positive CSF cytology, vitreous biopsy, or brain/leptomeningeal biopsy remain the current standard for diagnosis. A combined stepwise systematic approach outlined here may facilitate an expeditious, comprehensive presurgical evaluation for cases of suspected CNS lymphoma.

Intraspecific variability and genetic structure in Meloidogyne chitwoodi from the USA

Meloidogyne chitwoodi is a quarantine pathogen and a severe problem of potato. Intraspecific variation and genetic structure are not well characterised for M. chitwoodi, but are critical to avoid misidentification and to optimise management strategies. The objective of this study was to analyse the morphological and molecular variation of four M. chitwoodi isolates, representing all races and pathotypes currently known in the USA. Despite statistically significant morphological variation among adult females, morphometrics were not able reliably to distinguish M. chitwoodi isolates. In contrast to morphology, molecular traits that are determined by nuclear ribosomal genes were stable across all isolates. Malate dehydrogenase, esterase and superoxide dismutase isozyme phenotypes were conserved in all isolates, which is an important finding for diagnostics. To gain insight into the genetic structure of M. chitwoodi, we analysed a mitochondrial DNA segment including a partial region of COII, tRNA-His and 16S rRNA genes. Genetic structure was weak and marked by high haplotype and low nucleotide diversity. We found a high level of mitochondrial heteroplasmy in M. chitwoodi. Taken together, our results indicate that there is significant intraspecific morphological and molecular variation in M. chitwoodi. Consequences for resistance breeding in potato and directions for phylogeographic studies to trace the origin of M. chitwoodi are discussed.

First Report of Laurel Oak as a Host for the Pecan Root-Knot Nematode, Meloidogyne partityla, in Florida

Roots of laurel oak (Quercus laurifolia Michx.), member of the family Fagaceae, were found to be heavily galled by the pecan root-knot nematode, Meloidogyne partityla, in two separate home gardens between 2010 and 2012, in Alachua Co., FL. Distinct round galls were observed on secondary and tertiary roots. Internally, root-knot nematode females were clearly visible when the roots were thinly sliced and egg masses were seen protruding from the root surfaces. The nematode species identification was performed using morphology of the male stylet, selected characters of the second-stage juveniles (J2), female perineal patterns, and esterase (EST) and malate dehydrogenase (Mdh) isozyme phenotypes. Morphology of perineal patterns of females, body, stylet, and tail length of the J2 and males all matched those of the original description of M. partityla (2). A swollen deeply grooved rectum was observed in the J2. The male stylet had a blunt tip with a prominent thickening at the junction between the cone and shaft. The stylet knobs of males and females were bipartite, each incised by a deep medium longitudinal groove (2). The isozyme phenotypes (EST = Mp3; Mdh = N1a) were consistent with those previously reported for M. partityla from Florida (1). Mitochondrial DNA (mtDNA) (3) and ribosomal internal transcriber spacer (ITS) DNA (4) of females were amplified to further confirm the nematode species identification. The mtDNA amplification using the C2F3/1108 primer set (3) and the ITS amplification using a recently available M. partityla specific primer set (4) produced fragments of approximately 530 bp and 550 bp, respectively. These were consistent with those already reported for this nematode species. This first report of a plant host for the pecan root-knot nematode outside of the family Juglandaceae indicates that the nematode may have migrated from Quercus species to pecan trees during the period when orchards were being established in Florida. To our knowledge, this is the first report of the pecan root-knot nematode infecting laurel oak.