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.     

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).     

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.     

Identification of two cross-neutralizing linear epitopes within the L1 major capsid protein of human papillomaviruses

The neutralizing activities of polyclonal antibodies and monoclonal antibodies (MAbs) obtained by immunization of mice with L1 virus-like particles (VLPs) were investigated by using pseudovirion infectivity assays for human papillomavirus type 16 (HPV-16), HPV-31, HPV-33, HPV-45, HPV-58, and HPV-59 to obtain a better definition of cross-neutralization between high-risk HPVs. In this study, we confirmed and extended previous studies indicating that most genital HPV genotypes represent separate serotypes, and the results suggest that the classification of serotypes is similar to that of genotypes. In addition, three cross-neutralizing MAbs were identified (HPV-16.J4, HPV-16.I23, and HPV-33.E12). MAb HPV-16.J4 recognized a conserved linear epitope located within the FG loop of the L1 protein, and HPV-16.I23 recognized another located within the DE loop. The results suggested that reactivity of MAb HPV-16.I23 to L1 protein is lost when leucine 152 of the HPV-16 L1 protein is replaced by phenylalanine. This confirmed the existence of linear epitopes within the L1 protein that induce neutralizing antibodies, and this is the first evidence that such linear epitopes induce cross-neutralization. However, the cross-neutralization induced by L1 VLPs represents less than 1% of the neutralizing activity induced by the dominant conformational epitopes, and it is questionable whether this is sufficient to offer cross-protection in vivo.     

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.     

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     

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.     

Pentraxin 3 (PTX3) expression in allergic asthmatic airways: role in airway smooth muscle migration and chemokine production

Background

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 is produced by immune and structural cells. However, very little is known about the expression of PTX3 and its role in allergic asthma.

Objectives and Methods

We sought to determine the PTX3 expression in asthmatic airways and its function in human airway smooth muscle cells (HASMC). In vivo PTX3 expression in bronchial biopsies of mild, moderate and severe asthmatics was analyzed by immunohistochemistry. PTX3 mRNA and protein were measured by real-time RT-PCR and ELISA, respectively. Proliferation and migration were examined using ³H-thymidine incorporation, cell count and Boyden chamber assays.

Results

PTX3 immunoreactivity was increased in bronchial tissues of allergic asthmatics compared to healthy controls, and mainly localized in the smooth muscle bundle. PTX3 protein was expressed constitutively by HASMC and was significantly up-regulated by TNF, and IL-1β but not by Th2 (IL-4, IL-9, IL-13), Th1 (IFN-γ), or Th-17 (IL-17) cytokines. In vitro, HASMC released significantly higher levels of PTX3 at the baseline and upon TNF stimulation compared to airway epithelial cells (EC). Moreover, PTX3 induced CCL11/eotaxin-1 release whilst inhibited the fibroblast growth factor-2 (FGF-2)-driven HASMC chemotactic activity.

Conclusions

Our data provide the first evidence that PTX3 expression is increased in asthmatic airways. HASMC can both produce and respond to PTX3. PTX3 is a potent inhibitor of HASMC migration induced by FGF-2 and can upregulate CCL11/eotaxin-1 release. These results raise the possibility that PTX3 may play a dual role in allergic asthma.