The virD operon of Agrobacterium tumefaciens Ti plasmid encodes a DNA-relaxing enzyme.

The virD locus of Agrobacterium tumefaciens Ti plasmid encodes functions necessary for endonucleolytic cleavage of transferred DNA (T-DNA) prior to its transfer to plant cells. For the overproduction of the VIRD proteins in Escherichia coli a tac-virD operon fusion was constructed. A significant increase in the accumulation of VIRD proteins was observed in a lon protease-deficient E. coli host. The presence of an overlapping open reading frame (ORF) upstream of the VIRD1 coding sequence had a negative effect on VIRD1 production. A preparation containing VIRD proteins catalyzes the conversion of supercoiled (form I) DNA to relaxed (form IV) DNA. This activity is similar to that of a DNA topoisomerase. The relaxation activity lacks DNA sequence specificity, requires magnesium ion, and has no requirement for an energy source. Studies with plasmids that had lost defined DNA segments encompassing various virD coding regions showed that VIRD1 is the DNA-relaxing enzyme. In a density gradient centrifugation experiment, the DNA-relaxing activity sedimented as a 21-kDa polypeptide. Earlier studies of Jayaswal et al. [Jayaswal, R., Veluthambi, K., Gelvin, S. & Slightom, J. (1987) (J. Bacteriol. 169, 5035-5045] have shown that in E. coli VIRD2 alone is not sufficient for endonucleolytic cleavage of T-DNA and requires VIRD1 for its activity.     

Studies on Relative Toxicities of Six Insecticides on Epigeic Earthworm, Perionyx excavatus

The 96 h LC₅₀ values of six insecticides were determined on a non-target epigeic earthworm Perionyx excavatus under laboratory conditions. Cypermethrin was found most toxic to P. excavatus (LC₅₀-0.008 mg/kg), followed by endosulfan (LC₅₀-0.03 mg/kg), carbaryl (LC₅₀-6.07 mg/kg), chlorpyrifos (LC₅₀-7.3 mg/kg), aldicarb (LC₅₀-10.63 mg/kg) and monocrotophos (LC₅₀-13.04 mg/kg). When these LC₅₀ values were compared with their respective recommended agricultural doses, aldicarb and carbaryl appeared more dangerous than other pesticides because of their lower LC₅₀ values than their respective recommended agricultural dose. Mean lethal time to cause 50% mortality at recommended agricultural dose (LT₅₀) also indicated that aldicarb achieved the fastest LT₅₀ (26 h) followed by endosulfan (38 h) and carbaryl (44 h) indicating the danger of these pesticides to P. excavatus.     

Design of a bifunctional fusion protein for ovarian cancer drug delivery: single-chain anti-CA125 core-streptavidin fusion protein.

We have developed a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A single-chain antibody variable domain (scFv) that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain (core-streptavidin-VL-VH and VL-VH-core-streptavidin orientations) using recombinant DNA technology and then expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed in a shaker flask culture, extracted from the periplasmic soluble protein, and affinity purified using an IMAC column. The two distinct activities (biotin binding and anti-CA125) of the bfFp were demonstrated using ELISA, Western blot and confocal laser-scanning microscopy (CLSM). The ELISA method utilized human NIH OVCAR-3 cells along with biotinylated bovine serum albumin (B-BSA) or biotinylated liposomes, whereas, the Western blot involved probing with B-BSA. The CLSM study has shown specificity in binding to the OVCAR-3 cell-line. ELISA and Western blot studies have confirmed the bifunctional activity and specificity. In the presence of bfFp, there was enhanced binding of biotinylated antigen and liposome to OVCAR-3 cells. In contrast, the control EMT6 cells, which do not express the CA125 antigen, showed minimal binding of the bfFp. Consequently, bfFp based targeting of biotinylated therapeutic drugs, proteins, liposomes, or nanoparticles could be an alternative, convenient method to deliver effective therapy to ovarian cancer patients. Peritoneal infusion of the bfFp-therapeutic complex could also be effective in locally targeting the most common site of metastatic spread.     

Awareness of warning symptoms and risk factors of stroke in the general population and in survivors stroke.

Awareness among the general population of the risk factors and warning symptoms of stroke is essential for preventative purposes and for immediate effective treatment. The aim of the present study was to assess the awareness, among the general population and stroke survivors, of the risk factors and warning symptoms of stroke, to develop an educational strategy for its prevention and immediate effective treatment. Six hundred and sixty stroke patients (370 male, 290 female) and 4000 people from the general population who accompanied the patients (2800 male, 1200 female) were interviewed, using three sets of questionnaires, on the risk factors and warning symptoms of stroke. Poor knowledge or awareness of the risk factors and warning symptoms of stroke was found in both groups. Both groups suggested educational programs for stroke using printed information, audiovisual programs and community survey programs using simple and understandable information for the prevention and immediate effective treatment of stroke. Poor awareness of stroke contributes to a delay in the arrival of patients in hospital emergency departments for immediate effective treatment. Multifaceted programs regarding stroke, including printed information, audiovisual programs and stroke service programs, are advocated by both patients and the general population to improve stoke treatment and prevention.     

Exophytic gliomas of the spinal cord

Intradural extramedullary glial tumours of the spinal cord are rare. We report for such tumours arising from the dorsal cord. Myelography and operative findings were almost similar to that of an intradural neurofibroma. Surgical removal had resulted in rewarding neurological recovery. One of them had a recurrence after six years and was re-explored. Anterolateral attachment near the root entry zone suggests its origin probably from the spinal cord with an exophytic growth.