Malate Dehydrogenase Isoenzymes Research Articles

Genetic characterization of silkworm (Bombyx mori L.) strains (Lepidoptera: Bombicidae) with different geographical origin on the basis of isozyme markers

The aim of the present study was to assess genetics variations within and among strains with different geographical origin, belonging to Bulgarian germplasm bank of mulberry silkworm (Bombyx mori L.) and to establish their relationships using isoenzyme markers. Polyacrilamide gel electrophoresis (PAGE) was used to study the isoenzymes of nonspecific esterases (EST), malate dehydrogenase (MDH) and acid phosphatase (ACP) from haemolymph, phosphoglucomutase (PGM) and hexokinase (HK) from silk glands and alkaline phosphatase (ALP) from midgut of mulberry silkworm (Bombyx mori L.). Variability was evident in all of these enzyme systems among twelve strains from Bulgarian germplasm resources of B. mori. Total of nine loci were detected. All of them (100%) were polymorphic. “Null” alleles in four loci were determined. Intra- and inter-strain polymorphism was obtained. The degree of polymorphism ranged from 0% to 77.80%. Low levels of observed heterozygosity in comparison with the expected one have been calculated in almost all of strains as well as deviations from Hardy-Weinberg equilibrium in almost all analyzed loci, resulting of excess of homozygotes. The value of FST was 0.4903. The dendrogram constructed with the values of genetic distance manifests that Romanian strain Cislau Tokay is formed one main clade while the rest strains studied (from Bulgaria, Japan, China, Vietnam, Spain, Syria and Egypt) are formed the other clade with subclades. The genetic data of the tested strains from different geographical regions, would be used for identifying suitable parents for breeding programs with a view to yield improvement in this species with economic significance.

Regulation of Thiamine (Vitamin B1)-Dependent Metabolism in Mammals by p53.

Transcriptional factor p53 is a master regulator of energy metabolism. Energy metabolism strongly depends on thiamine (vitamin B1) and/or its natural derivatives. Thiamine diphosphate (ThDP), which is a major thiamine derivative, affects p53 binding to DNA. In order to elucidate the mechanism of regulation of thiamine-dependent metabolism by p53, we assessed putative p53-binding sites near transcription starting points in genes coding for transporters and enzymes, whose function is associated with thiamine and/or its derivatives. The predictions were validated by studying cell metabolic response to the p53 inducer cisplatin. Expression of p53 and its known target, p21, has been evaluated in cisplatin-treated and control human lung adenocarcinoma A549 cells that possess functional p53 pathway. We also investigated the activity of enzymes involved in the thiamine-dependent energy metabolism. Along with upregulating the expression of p53 and p21, cisplatin affected the activities of metabolic enzymes, whose genes were predicted as carrying the p53-binding sites. The activity of glutamate dehydrogenase GDH2 isoenzyme strongly decreased, while the activities of NADP+-dependent isocitrate dehydrogenase (IDH) and malic enzymes, as well as the activity of 2-oxoglutarate dehydrogenase complex at its endogenous ThDP level, were elevated. Simultaneously, the activities of NAD+-dependent IDH, mitochondrial aspartate aminotransferase, and two malate dehydrogenase isoenzymes, whose genes were not predicted to have the p53-binding sequences near the transcription starting points, were upregulated by cisplatin. The p53-dependent regulation of the assayed metabolic enzymes correlated with induction of p21 by p53 rather than induction of p53 itself.

Regulation of Malate Dehydrogenases and Glutamate Dehydrogenase of Mammalian Brain by Thiamine in vitro and in vivo.

To study the mechanisms of the non-coenzyme action of thiamine and its diphosphate (ThDP) on brain proteins, proteins of acetone extract of bovine brain synaptosomes or the homogenate of rat brain cortex were subjected to affinity chromatography on thiamine-modified Sepharose. In the step-wise eluates by thiamine (at pH 7.4 or 5.6), NaCl, and urea, the occurrence of glutamate dehydrogenase (GDH) and isoenzymes of malate dehydrogenase (MDH) along with the influence of thiamine and/or ThDP on the enzymatic activities were characterized using mass spectrometry and kinetic experiments. Maximal activation of the malate dehydrogenase reaction by thiamine is observed after the protein elution with the acidic thiamine solution, which does not elute the MDH1 isoenzyme. Effects of exogenous thiamine or ThDP on the GDH activity may depend on endogenous enzyme regulators. For example, thiamine and/or ThDP activate the brain GDH in eluates from thiamine-Sepharose but inhibit the enzyme in the crude preparations applied to the sorbent. Inhibition of GDH by ThDP is observed using the ADP-activated enzyme. Compared to the affinity chromatography employing the elution by thiamine at pH 7.4, the procedure at pH 5.6 decreases the activation of GDH by thiamine (but not ThDP) in the eluates with NaCl and urea. Simultaneously, the MDH2 content and total GDH activity are higher after the affinity elution at pH 5.6 than at pH 7.4, suggesting the role of the known interaction of GDH with MDH2 in stabilizing the activity of GDH and in the regulation of GDH by thiamine. The biological potential of thiamine-dependent regulation of the brain GDH is confirmed in vivo by demonstration of changes in regulatory properties of GDH after administration of a high dose of thiamine to rats. Bioinformatics analysis of the thiamine-eluted brain proteins shows a specific enrichment of their annotation terms with "phosphoprotein", "acetylation", and "methylation". The relationship between thiamine and the posttranslational modifications in brain may contribute to the neuroprotective effects of high doses of thiamine, including the regulation of oxidation of the major excitatory neurotransmitter in brain - glutamate.

Molecular Characterization of Twenty-Five Marine Cyanobacteria Isolated from Coastal Regions of Ireland.

Twenty-five marine cyanobacteria isolated from Irish coasts were characterized based on their morphological characters and 16S rRNA gene sequence analysis. In addition, superoxide dismutase (SOD) and malate dehydrogenase (MDH) isoenzyme banding patterns were used to differentiate two morphologically ambiguous isolates. In this study, six new cyanobacteria-specific primers were designed, and a 16S rRNA gene of twenty-five morphologically diverse cyanobacteria was successfully PCR amplified (1198–1396 bps). Assembled 16S rRNA sequences were used both for a basic local alignment search tool (BLAST) analysis for genus-level identification and to generate a phylogenetic tree, which yielded two major clusters: One with morphologically homogenous cyanobacteria and the other with morphologically very diverse cyanobacteria. Kamptonema okenii and Tychonema decoloratum were isolated from a single field sample of Ballybunion and were originally identified as the same ‘Oscillatoria sp.’ based on preliminary morphological observations. However, an alignment of 16S rRNA gene sequences and SOD and MDH isoenzyme banding pattern analyses helped in differentiating the morphologically-indistinguishable ‘Oscillatoria sp.’. Finally, after a re-evaluation of their morphological characters using modern taxonomic publications, the originally identified ‘Oscillatoria sp.’ were re-identified as Kamptonema okenii and Tychonema decoloratum, thus supporting the polyphasic approach of cyanobacteria characterization.

Действие двухвалентных катионов металлов на функционирование пероксисомальной малатдегидрогеназы из листьев кукурузы

Модифицированная многостадийная очистка, включающая в себя изоплотностное центрифугирование с помощью ступенчатого градиента сахарозы при концентрации от 1,3 до 2,5 М. Разделение субклеточных фракций осуществляли путем ультрацентрифугирования при 100 000 g. Определяющее значение для эффективной очистки фермента имела ионообменная хроматография, которая позволила получить гомогенные препараты малатдегидрогеназы из мезофилла листьев кукурузы. Степень очистки фермента, обладающего удельной активностью 513 Е/мг белка, составила 135 раза, а выход - 4%. На полученном препарате было исследовано влияние двухвалентных катионов, таких как кальций, барий, магний и марганец. Графоаналитическим методом были определены тип и характер регулирующего действия исследуемых катионов на малатдегидрогеназную систему. Показано, что ионы магния в малых дозах (до 2мМ) оказывают активирующее действие на работу энзима, а в больших (более 2 мМ) – ингибируют активность малатдегидрогеназы. Другие исследованные катионы оказывали ингибирующее действие на функционирование фермента

Purification and characterization of malate dehydrogenase from sheep liver (Ovis aries): Application in AST assay diagnostic kit

Malate dehydrogenase enzyme is a major component in aspartate aminotransferase (AST) diagnostic kit that used in diagnosis and monitoring of liver diseases. This study aimed at purification and characterization of MDH enzyme from sheep liver for direct application in preparation of AST kit. Two malate dehydrogenase isoenzymes were resolved from sheep liver by chromatography on DEAE-cellulose column, one major sheep liver MDH (SLMDH) and another minor one. SLMDH was extracted and purified by ammonium sulfate fractionation and chromatographical separation on anion exchanger and gel filtration resins. The purified sheep liver MDH specific activity is 6.7 units / mg protein representing 11.6 purification folds and 27.7 % yield. The molecular mass of SLMDH intact protein is 68 ± 1.6 kDa. SLMDH turned out to be homogeneous and consists of two identical subunits of 34 ± 1.2 kDa each. The SLMDH isoelectric point (pI) value is estimated at pH 6.2 and displayed its optimum activity at pH 9.6 and has Km value of 1.4 mM for NAD and 11 mM for malate. FeCl2, NiCl2 and ZnCl2 inhibited the SLMDH activity. The purified SLMDH is applied in the preparation of AST diagnostic kit that found sensitive and comparable with a commercially available one.

Characterization of the Kinetics of Cardiac Cytosolic Malate Dehydrogenase and Comparative Analysis of Cytosolic and Mitochondrial Isoforms

Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.

Induction and resistance against Fusarium wilt disease of tomato by using sweet basil (Ocimum basilicum L) extract

Akladious, S. A., Isaac, G. S. and Abu-Tahon, M. A. 2015. Induction and resistance against Fusarium wilt disease of tomato by using sweet basil (Ocimum basilicum L) extract. Can. J. Plant Sci. 95: 689–701. The antifungal activity of Ocimum basilicum (sweet basil) extract against Fusarium oxysporum f. sp. lycopersici race 3 the causal agent of tomato wilt and its ability in inducing disease resistance were studied in vivo using seed-soaking treatment before sowing. Plants were harvested at 45 and 105 d (vegetative and flowering stages) after sowing. Treatment with O. basilicum extract decreased the disease incidence from 94.70 to 18.00%. Results revealed that growth parameters and photosynthetic pigments were markedly inhibited in tomato plants in response to Fusarium wilt disease, whereas the contents of non-enzymatic and enzymatic antioxidants were increased as compared with healthy control plants. Moreover, presoaking in basil extract enhanced all the mentioned parameters in both healthy and infected plants. SDS-PAGE analysis of tomato leaves revealed that seed treated with basil extract resulted in an induction of novel protein bands during the vegetative stage. These new proteins were not detected in untreated healthy or infected control plants. Electrophoretic studies of polyphenol oxidase, esterase and malate dehydrogenase isoenzymes showed wide variations in their intensities and densities among all treatments. It seems that O. basilicum extract was able to enhance the biological control of Fusarium wilt disease of tomato.

The intracellular localization of malate dehydrogenase isoenzymes in Pisum arvense roots

Mitochondria and plastids were isolated from <em>Pisum arvense</em> root cells by sucrose density gradient centrifugation. The individual subcellular fractions so obtained were subjected to isoelectric focusing on cellulose acetate strips. Mitochondria and plastids each contained one NAD -malate dehydrogenase, while three isoenzymes were associated with the supernatant.

Establishment and Biological characterization of a Large Yorkshire Pig fibroblast line

Aim: This study aimed to establish the fibroblast cell bank of the Large Yorkshire pig in the form of somatic cells, and to study its biological characteristics, to explore long-term conservation and sustainable use of this genetic resource. Material and methods: Ear marginal tissue of the Large Yorkshire pig was used to establish a fibroblast cell line by direct culture of explants and cryopreservation techniques, and the quality of the cell line was assessed. Results: Biological analyses showed that the cells were morphologically homogenous fibroblasts, and the estimated population doubling time (PDT) was 72 hours. The growth curve appeared as a typical S shape as the cell growth passed through a latent phase, a logarithmic phase and a plateau phase. Tests for bacteria, fungi, viruses and Mycoplasma were negative. Analysis of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzymes ruled out cross-contamination between cell lines. Karyotyping and G-banding indicated a total chromosome number of 2n = 36; the proportion of diploid cells in the population was 91-96%. The transfection efficiencies of pEGFP-N3, pEYFP-N1 and pDsRed1-N1 were between 5.14 and 12.13%. Bioindicators of the cell bank met all the standards of the American Type Culture Collection (ATCC). Conclusion: This study not only shows the effective preservation of the Large Yorkshire pig germplasm at the cellular level, but also provides a valuable experimental resource for fields including cell biology, medicine, genomics, post-genomics, genetic engineering and embryo engineering.

Effects of NaCl on growth and activity of enzymes involved in carbon metabolism in leaves of tobacco (Nicotiana rustica)

The adverse effects of salt should not be the same in tobacco plants exposed to a permanent and transient high concentration of NaCl in its environment. Experiments were conducted in order to verify the hypothesis of reversibility of NaCl effects. The study of this reversibility is checked by monitoring a number of parameters in pre-stressed plants and then, replaced in normal conditions. Plants previously grown for 30 days on basic medium were treated for 7 days with 200 mM NaCl and then placed back on the basic culture without NaCl for 10 days. The results show that NaCl suppression leads to a resumption of growth with a decrease in the concentration of sodium (Na + ) and chloride ions (Cl - ). Hence, potassium content (K + ) increases gradually in the leaves to reach the level obtained with unstressed plants. At the same time, there is a stimulation of the activities of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase isoenzymes (NAD, NADP, NADH and NADPH-MDH) and isocitrate dehydrogenase (ICDH) after NaCl had been removed. Along with the boosting of the activity of these enzymes involved in the process of carbon assimilation, there is a gradual decrease in soluble sugars content, suggesting a resumption of the normal activity of photosynthetic assimilation process. All these results verify our hypothesis and can be explained by the ability of the plant to dilute the effects of Na + and Cl - during the recovering period. An important result of this study is that a transient salinity is not necessarily followed by a significant depreciation in product yield or quality. Keywords: Tobacco, NaCl, reversibility, phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), isocitrate dehydrogenase (ICDH)

Tissue-specific isoenzyme variations in Arabian camel, <i>Camelus dromedaries</i>

Malate dehydrogenase, α-esterase and β-esterase isoenzymes have been analyzed by native polyacryla-mide gel electrophoresis in different tissues (liver, kidney, muscle and hump) of the Arabian Camel, Camelus dromedaries in order to study the tissue specificity of these isoenzymes. Malate dehy-drogenase recorded three fractions while both este-rases recorded two fractions in different studied tissues. Malate dehydrogenase was considered as a key isoenzyme in discriminating among the different studied tissues. The percentage amount of both Mdh-1 and Mdh-2 showed significant difference among the different tissues while Mdh-3 recorded significant variation between muscle and hump tissues. The significant variation in the amount of Mdh isoenzyme (on the level of either total or fractions) among the different studied tissues may reflect the role of this enzyme for energy production in this desert animal. With respect to esterases, the percentage amount for both fractions and the total enzyme did not show significant difference among different tissues except β-Est-2 which was significantly higher in hump than in muscle. This enzyme showed higher thickness and intensity in both liver and kidney than in the muscle and hump indicating its greater activity in both liver and kidney.

Tissue-Specific Isoenzyme Variations in Tilapia Fish, <i>Oreochromis niloticus</i>

Native polyacrylamide gel electrophoresis have been used to analyze malate dehydrogenase (MDH), acid phosphatase (Acph) and peroxidase (Px) isoenzymes in different tissues (liver, kidney, muscle and heart) of the tilapia fish, Oreo- chromis niloticus in order to study the tissue specificity of these isoenzymes. Three, two and one fractions have been recorded respectively for the three isoenzymes in different studied tissues. The MDH-1 and MDH-2 have been ex- pressed only in muscle and heart while MDH-3 has been expressed in all studied tissues. The percentage amount of MDH in general varied significantly between muscle and different studied tissues. With respect to acid phosphatase, the percentage amount of the total enzyme showed significant difference between liver and muscle and that this variation may be due to higher gene activity in liver. Peroxidase isoenzyme was recorded in liver and heart only with significant increase in liver. The kidney was the least among the studied tissues in showing gene expression for the studied isoenzymes and therefore, liver, heart and muscle tissues are better applicable in studying the isoenzymatic profiles for fish physiology and systematics.

Establishment and biological characteristics of a Jingning chicken embryonic fibroblast bank

Using tissue explantation and cryopreservation biotechniques, a Jingning chicken embryonic fibroblast bank was successfully established, which includes 43 embryo samples and a stock of 178 cryovials, each one containing 3.0×106 cells. Most of the cells were apparently fibroblasts in their morphology, and the population doubling time (PDT) was about 48 h. The total chromosome number of a diploid cell was 78. According to karyotyping and G-banding, the diploid rate in the cell bank was 97.62±2.12%. The cells were tested for microbial contamination and found free of infections from bacteria, fungi, viruses and mycoplasms. There was no cross-contamination from other cell lines as revealed by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzyme polymorphisms. Six fluorescent proteins were transfected into the Jingning chicken embryonic fibroblasts, and the transfection efficiencies of these genes were between 10.1 and 41.9%. All the tests showed that the quality of the cell line conforms to the quality criteria of the ATCC (American type culture collection). This work succeeded not only in preserving the genetic resources of Jingning chicken, but it also established a new protocol to preserve endangered animal breeds.

Establishment and characteristics of white ear lobe chicken embryo fibroblast line and expression of six fluorescent proteins in the cells

A white ear lobe chicken embryo (WELCE) fibroblast cell bank, containing 322 tubes of frozen cells, was successfully established from primary explants of 57 embryo samples. The cells were morphologically consistent with fibroblasts, and the growth curve was sigmoidal with a population doubling time (PDT) of 48 h. Karyotyping and G-banding indicated a total chromosome number of 2n=78; the rate of diploidy in the cell bank was 97.62%. The cells were also free from bacterial, fungal, viral and mycoplasma contamination. Analysis of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzymes ruled out cross-contamination between cells. In order to study exogenous gene expression, six fluorescent proteins were transfected into the WELCE cells. The transfection efficiency of these genes was between 10.1 and 41.9%. The corresponding fluorescence was distributed throughout the cytoplasm and nucleus 24h after transfection. The results indicate that the quality of the cell line meet the quality requirements of the ATCC (American Type Culture Collection).

Characterization of a stable isoenzyme of malate dehydrogenase (MDH1) from blood stream Trypanosoma vivax

MDH1, isoenzyme of malate dehydrogenase from blood stream Trypanosoma vivax was characterized. The enzyme was active over a broad pH and temperature range with optimal values of 5.0 and 35oC respectively. The energy of activation was 20.58kj/mol and the pKa values were 6.8 and 7.8 implicating ionizable groups at the catalytic site. Kinetic studies conducted in the direction of oxaloacetate reduction gave KM of 0.56 and 0.3 mM and Vmax of 4.6 and 3.2 µmol./ min/mg for oxaloacetate and NADH respectively. Similarly, in the reverse reaction, malate had KM of 0.16 Mm and Vmax of 57 µmol./min/mg, and KM and Vmax of NAD+ were 0.1 5 mM and 48 µmol./min/mg. The enzyme was inhibited competitively with NAD+ as a product inhibitor and uncompetitive with malate. The product inhibition studies suggest bi-bi ordered sequential mechanism of catalysis. Some TCA cycle intermediates also inhibited to different extent the activity of the enzyme. Key words: Malate dehydrogenase, Trypanosoma vivax, isoenzyme.

ANALISIS ISOENZIM UNTUK MEMPELAJARI VARIASI GENETIK SAPI BALI DI PROVINSI BALI

The aim of this research was to know Bali cattle genetic variation according to the band pattern of isoenzyme. Esteraseand Malate dehydrogenase Isoenzymes of Bali cattle were studied. Using the native-PAGE method, the analysis has been made of the genetic structure and variation of the bali cattle population. Isoenzyme isolated from leucocytes cell using homogenation method by adding Phosphat Buffer Saline (PBS). A hundred sample of Bali cattles were taken in Mambang, Slemadeg and Kuwumkeladi. The result of this research indicate that from the 2 different enzyme, 3 loci were detected, and 1 of them was polymorphic (MDH-2). There was null allele phenomenom in MDH-2 locus. The loci polymorphic proportion of three population were 0,333. Chi-Square analysis of three population were 1.251–1.560. The heterozygosity value of three population (Mambang, Slemadeg and Kuwumkeladi) were 0.098, 0.111 and 0.118, respectively.

ANALISIS ISOENZIM UNTUK MEMPELAJARI VARIASI GENETIK SAPI BALI DI PROVINSI BALI

The aim of this research was to know Bali cattle genetic variation according to the band pattern of isoenzyme. Esteraseand Malate dehydrogenase Isoenzymes of Bali cattle were studied. Using the native-PAGE method, the analysis has been made of the genetic structure and variation of the bali cattle population. Isoenzyme isolated from leucocytes cell using homogenation method by adding Phosphat Buffer Saline (PBS). A hundred sample of Bali cattles were taken in Mambang, Slemadeg and Kuwumkeladi. The result of this research indicate that from the 2 different enzyme, 3 loci were detected, and 1 of them was polymorphic (MDH-2). There was null allele phenomenom in MDH-2 locus. The loci polymorphic proportion of three population were 0,333. Chi-Square analysis of three population were 1.251–1.560. The heterozygosity value of three population (Mambang, Slemadeg and Kuwumkeladi) were 0.098, 0.111 and 0.118, respectively.

Comparison of properties of malate dehydrogenase isoenzymes from hare and rabbit hearts

The hare heart mitochondrial malate dehydrogenase (mMDH) was established to have a much higher electrophoretic mobility than the corresponding enzyme from the rabbit heart. Differences of kinetic properties of both mMDH and cytoplasmic malate dehydrogenase (cMDH) from these two sources were shown. The hare heart mMDH and cMDH isoenzymes have a higher affinity to malate (in direct reaction) and oxaloacetate and NADH (in reverse reaction), i.e., they have lower K M values in comparison with the isoenzymes from the rabbit heart. Malate dehydrogenase seems to operate more effectively in the hare heart, which might be important in adaptive and evolutionary aspects.

Production of potato intraspecific somatic hybrids with improved tolerance to PVY and Pythium aphanidermatum

Somatic hybridization can be an interesting alternative for the selection of heterozygous and vigorous potato plants through combination of dihaploid genomes. The resulting hybrids can harbour interesting characters and thus can be used in agriculture if they are in agreement with agronomic criteria. In this report, we used an intraspecific somatic hybridization technique for the production of tetraploid potato lines. Two parental combinations were used in protoplast electrofusion procedure: Aminca-Cardinal and Cardinal-Nicola. The selection of somatic hybrids was based on in vitro plant vigour. Therefore, among the 75 regenerated plants obtained from Aminca-Cardinal fusion, 3 putative hybrids were retained and 2 plant lines were selected among the 54 regenerated from the Cardinal-Nicola fusion. Heterosis was observed in the larger hybrid tuber size compared to the parents'. Our results also showed a precocity in the in vitro tuberization for the hybrids. Moreover, all of the regenerated putative hybrids were tetraploid (2n=4x=48 chromosomes). Isocitrate dehydrogenase and malate dehydrogenase isoenzyme analyses confirmed the hybrid nature of these lines. A molecular characterization performed by PCR amplification of simple sequence repeats and inter-simple sequence repeats confirmed that all these lines were somatic hybrids. The effect of potato virus Y infection on these hybrid lines was tested by mechanical inoculation of plants cultivated in a greenhouse. The majority displayed a reduction of infection rate associated with a delayed appearance of symptoms compared to the parents. Moreover, complete resistance was noted for one hybrid line (CN2). All hybrids also showed improved tolerance to Pythium aphanidermatum infection during tuber storage or after plant inoculation.