Purification and characterization of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the dromedary camel

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12),a key enzyme ofcarbon metabolism,was purified and characterized to homogeneity from skeletal muscle of Camelusdromedarius.The protein was purified approximately 26.8 folds by conventional ammonium sulphatefractionation followed by Blue Sepharose CL-6B chromatography,and its physical and kinetic propertieswere investigated.The native protein is a homotetramer with an apparent molecular weight of approximately146 kDa.Isoelectric focusing analysis showed the presence of only one GAPDH isoform with an isoelectricpoint of 7.2.The optimum pH of the purified enzyme was 7.8.Studies on the effect of temperature onenzyme activity revealed an optimal value of approximately 28-32 ℃ with activation energy of 4.9 kcal/mol.The apparent K_m values for NAD~ and DL-glyceraldehyde-3-phophate were estimated to be 0.025±0.040mM and 0.21±0.08 mM, respectively. The V_(max) of the purified protein was estimated to be 52.7±5.9 U/mg.These kinetic parameter values were different from those described previously, reflecting protein differencesbetween species.     

Partial Resistance of Carrot to Alternaria dauci Correlates with In Vitro Cultured Carrot Cell Resistance to Fungal Exudates

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.     

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand²-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect. Received: 30 December 1997 / Accepted: 21 February 1998     

A 660-Kb Deletion with Antagonistic Effects on Fertility and Milk Production Segregates at High Frequency in Nordic Red Cattle: Additional Evidence for the Common Occurrence of Balancing Selection in Livestock

In dairy cattle, the widespread use of artificial insemination has resulted in increased selection intensity, which has led to spectacular increase in productivity. However, cow fertility has concomitantly severely declined. It is generally assumed that this reduction is primarily due to the negative energy balance of high-producing cows at the peak of lactation. We herein describe the fine-mapping of a major fertility QTL in Nordic Red cattle, and identify a 660-kb deletion encompassing four genes as the causative variant. We show that the deletion is a recessive embryonically lethal mutation. This probably results from the loss of RNASEH2B, which is known to cause embryonic death in mice. Despite its dramatic effect on fertility, 13%, 23% and 32% of the animals carry the deletion in Danish, Swedish and Finnish Red Cattle, respectively. To explain this, we searched for favorable effects on other traits and found that the deletion has strong positive effects on milk yield. This study demonstrates that embryonic lethal mutations account for a non-negligible fraction of the decline in fertility of domestic cattle, and that associated positive effects on milk yield may account for part of the negative genetic correlation. Our study adds to the evidence that structural variants contribute to animal phenotypic variation, and that balancing selection might be more common in livestock species than previously appreciated.     

Biodegradation of polyphenols with immobilized Candida tropicalis under metabolic induction

During olive oil production, large quantities of olive mill wastewater (OMW) are produced. This wastewater material, containing a high level of phenolic compounds, poses a serious environmental problem in almost all Mediterranean countries. Candida tropicalis YMEC14 was used as an extremophile strain to design an aerobic biotreatment process to detoxify OMW and reduce its polluting organic load. The process was enhanced by directing yeast metabolism towards biodegradation pathways using hexadecane as co-metabolite and by immobilizing yeast cells in calcium alginate beads. Under immobilization conditions, C. tropicalis YMEC14 grown at 40 degrees C in OMW supplemented with hexadecane resulted in 69.7%, 69.2% and 55.3% reduction of chemical oxygen demand, monophenols and polyphenols, respectively, after a 24-h fermentation cycle.     

Functional expression of IL-9 receptor by human neutrophils from asthmatic donors: role in IL-8 release

Human polymorphonuclear neutrophils (PMNs) express surface receptors for various inflammatory mediators, including IgE and IL-4. Recently, the IL-9R locus has been genetically linked to asthma and bronchial hyperresponsiveness in humans. In this study, we evaluated expression of the IL-9R and the effect of IL-9 on human PMNs. RT-PCR analysis showed the presence of IL-9Ralpha-chain mRNA in PMN RNA preparations from asthmatic patients. Using FACS analysis, surface expression of IL-9Ralpha was detected on PMNs freshly isolated from asthmatics, and to a lesser extent on normal controls. In addition, protein expression of IL-9Ralpha was also detected in peripheral blood and bronchoalveolar lavage PMNs. Furthermore, functional studies showed that IL-9 stimulation of PMNs results in the release of IL-8 in a concentration-dependent manner. The anti-IL-9 neutralizing Ab suppressed this effect, but had no effect on GM-CSF-induced IL-8 release from PMNs. Taken together, these findings suggest a novel role for PMNs in allergic disease through the expression and activation of the IL-9R.     

Toxicity of hexachlorobenzene in Meriones unguiculatus: effects on thyroid and liver

The effect of in vivo administered hexachlorobenzene (HCB) on liver and thyroid was studied on Meriones unguiculatus. HCB (1.6, 4, and 16 mg/kg of body weight) has been administered orally to meriones for 30 days. At the end of the experiment, the body weight of the animals did not show significant change. However, the higher dose of HCB treatment led to a pronounced hepatic hypertrophy comparatively to controls. Histological observations revealed many cytomorphological alterations. Cellular necrosis, periportal, and centrolobular vein congestion and cytoplasmic vacuolisation were noted and correlated with the administered doses of HCB. The higher dose of HCB induced modifications in the activities of hepatic transaminases and on thyroid hormones levels: ALAT activity level was more pronounced in males (170+/-24.7 U/l vs. 52.66+/-8.29 U/l in controls) than in females (120+/-12.47 U/l vs. 56+/-5 U/l in controls). However, ASAT activity increased significantly only in females (259+/-29 U/l vs. 244.66+/-18 U/l in controls). Plasma total triiodothyronine (TT3) and total thyroxine (TT4) levels seemed to be sex-dependent in intoxicated animals, since TT4 decreased significantly in males (21.95+/-7.46 nmol/l vs. 40.59+/-1.08 nmol/l in controls) and TT3 in females (1.42+/-0.11 nmol/l vs. 3.96+/-0.48 nmol/l in controls).     

Inositol polyphosphate 4-phosphatase B as a regulator of bone mass in mice and humans

Osteoporosis is a multifactorial genetic disease characterized by reduction of bone mass due to dysregulation of osteoclast differentiation or maturation. Herein, we identified a regulator of osteoclastogenesis, the murine homolog of inositol polyphosphate 4-phosphatase type IIα (Inpp4bα). Expression of Inpp4bα is detected from early osteoclast differentiation to activation stage. Targeted expression of native Inpp4bα ex?vivo repressed whereas phosphatase-inactive Inpp4bα stimulated osteoclast differentiation. Inpp4bα acts on intracellular calcium level that modulates NFATc1 nuclear translocation and activation. In?vivo mice deficient in Inpp4b displayed increased osteoclast differentiation rate and potential resulting in decreased bone mass and osteoporosis. Importantly, INPP4B in human was identified as a susceptibility locus for osteoporosis. This study defined Inpp4b as a major modulator of the osteoclast differentiation and as a gene linked to variability of bone mineral density in mice and humans.