We investigated the molecular response of degenerated human cervical and lumbar nucleus pulposus (NP) cells following cytokine treatment. Degenerated cervical and lumbar discs (8 each) were obtained from patients who underwent discectomy for degenerative disc disease; NP cells were isolated and cultured. The mRNA expressions of aggrecan, alkaline phosphatase, type I collagen, type II collagen, osteocalcin, and Sox9 in the 2 groups were compared by real-time PCR, before and following treatment with rhBMP-2 and TGF-β1. Immunoreactivity was analyzed to check protein activity.
The molecular and biochemical effects of an insecticidal toxin extracted from Meloidae beetles were investigated on Helicoverpa armigera. The toxin was identified as cantharidin, a well-known natural compound produced by beetles of family Meloidae and Oedemeridae. Furthermore, the effect of the toxin on the metabolic enzymes alkaline phosphatase (ALP) and glutathione S-transferase (GST), responsible for the metabolism of insecticides, was also investigated.
Alkaline phosphatase (ALP) of Helicoverpa armigera Hub. (Lepidoptera; Noctuidae) (GenBank accession No. EU729322) was cloned and expressed. The target gene H.a-ALP, having an open reading frame of 1608 bp, was reverse-transcribed from cDNA by the polymerase chain reaction. The open reading frame of the target gene was cloned into the pET-32a expression vector to obtain recombinant protein in Escherichia coli DE-3 cells for the subsequent production of polyclonal antibody. New Zealand white rabbits were used for production of anti-pET-32a-H.a-ALP.
The present study was designed to identify alkaline phosphatases in non-permeabilized hyphal cells of the fungus Neurospora crassa by staining these enzymatic activities with a modified azo dye coupling method. Our strategy allowed the identification of three non-specific alkaline phosphatase activities, one of them possibly being a novel putative enzyme, which is not responsive to either Mg2+ or EDTA.