Biochemical Characterization of Molybdenum-Cofactor in a Cyanobacterium, Nostoc muscorum

Cell-free extracts of nitrate-grown Nostoc muscorum containnitrate reductase and molybdenum-cofactor activities. Whilenitrate reductase activity is associated with the paniculatefraction, cofactor activity is found predominantly in the solublefraction. This activity was distributed between two pools. Inone pool, the molybdenumcofactor is associated with a carrier(protein) of approximately 30,000 Da with an S_{20, w} between2.3 and 2.5. The carrier-bound cofactor is non-dialyzable andis found along with the major proteins during filtration inSephadex G-25 and G-100. The second pool contains free or unboundcofactor. It is separated from soluble proteins by dialysiswith a membrane with a pore-size of 10 to 15 kDa. However, itis retained with a membrane with a pore size of 1 kDa. It isin the included volume during chromatography through SephadexG-25. Its molecular mass is estimated to be between 1,000 and5,000 Da. The molybdenum content was proportional to cofactoractivity in both pools. Reducing agents increased cofactor activity.However, activity in both pools was sensitive to heat, acid,and oxidative treatments. The carrier protein appears to givesome protection. ¹Fulbright Scholar from Department of Biological Sciences, R.D. University, Jabalpur-482001, India. To whom reprint requestsshould be addressed. (Received June 22, 1987; Accepted August 21, 1987)     

Naturally occurring anti-band 3 autoantibodies recognize a high molecular weight protein on the surface of Plasmodium falciparum infected erythrocytes

Naturally occurring anti-band 3 autoantibodies bind to erythrocytes infected with a knobby variant of the human malaria Plasmodium falciparum (FCR-3 strain). The autoantibodies recognized a greater than 240 kDa protein in SDS extracts made from surface iodinated infected erythrocytes. The antigen was associated only with erythrocytes infected with a knobby variant, and was removed by trypsin treatment of intact infected cells. By two-dimensional peptide map analysis the antigen was shown to be structurally related to the human erythrocyte anion transporter, band 3.     

Qa-2 antigen encoded by Q7b is biochemically indistinguishable from Qa-2 expressed on the surface of C57BL/10 mouse spleen cells

Qa-2 was immunoprecipitated from the surface of 125I-labeled C57BL/10 (B10) mouse spleen cells and compared with Qa-2 immunoprecipitated from the surface of R1.1 thymoma cells transfected with Q7b. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that Qa-2 glycoproteins from both of these sources have a relative molecular mass of approximately 37 kDa. After treatment with endoglycosidase F, the Qa-2 polypeptide chains derived from C57BL/10 spleen and Q7b-transfected R1.1 cells displayed identical mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis because of removal of N-linked oligosaccharide residues. Furthermore, treatment of Qa-2 proteins from both sources with cyanogen bromide or alpha-chymotrypsin resulted in identical peptide fragmentation patterns. These results therefore provide a biochemical correlation between a cloned Qa-region gene produce expressed on the surface of transfected cells, and the Qa-2 glycoprotein on spleen cells that was described a decade ago by serologic methods.     

Induction of circulating phospholipase A(2) by intravenous administration of recombinant human tumour necrosis factor

We have examined the effects of intravenous infusion of recombinant human tumour necrosis factor (rh-TNF) on serum activity of phospholipase A(2) (PLA(2)) in patients with malignancies. Nine patients received a 24 h continuous intravenous infusion ranging from 1.0 x 10(5) U/m(2) to 3.0 x 10(5) U/m(2); 14 patients received a 5 day continuous intravenous infusion ranging from 0.5 x 10(5) U/m(2)/day to 3.0 10(5) U/m(2)/day. Twenty one of 23 patients responded with marked increases in serum PLA(2) activity that were detectable 3 h after the beginning of the rh-TNF infusion and reached maximum levels at 18 h with a mean increase of 16.2-fold. In patients receiving a 5 day rh-TNF infusion, the highest levels of PLA(2) were observed after the first day of infusion. Serum PLA(2) activity declined continuously to 2.9-fold above baseline at the end of the infusion. A significant correlation was noted between the dose of infused rh-TNF and the maximum increase in PLA(2) activity. To our knowledge, this is the first time that an association between intravenous TNF administration and induction of circulating PLA(2) in man has been established.     

Comparative testing of disinfectant and antiseptic products using proposed European suspension testing methods

The development of standard suspension test methods for disinfectants and antiseptics for adoption in Europe is described. Evaluation of a range of disinfectant agents and products currently used in the UK under conditions as proposed for these tests indicates that the majority of products diluted in water of standard hardness showed satisfactory activity producing a 4·5–5 log reduction in viable count within 5 min against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Proteus mirabilis and Candida albicans in the absence and presence of 1% albumin. All the products, when diluted with distilled water, produced greater than 5 log reduction in 60 min.     

Calcium at the surface of cardiac plasma membrane vesicles: Cation binding,surface charge screening,and Na−Ca exchange

Summary Calcium binding and Na–Ca exchange activity were measured in isolated cardiac plasma membrane vesicles under various ionic conditions. A model was developed to describe the Ca binding characteristics of cardiac sarcolemmal vesicles using the Gouy-Chapman theory of the diffuse double layer with specific cation binding to phospholipid carboxyl and phosphate groups. The surface association constants used for Ca, Na, K and H binding to both of these groups were 7, 0.63, 0.3 and 3800m ^–1, respectively. This model allows the estimation of surface [Ca] under any specific ionic conditions. The effects of the divalent screening cation, dimethonium, on Ca binding and Na–Ca exchange were compared. Dimethonium had no significant effect on Ca binding at high ionic strength (150mm KCl), but strongly depressed Ca binding at low ionic strength. Dimethonium had no significant effect on Na–Ca exchange (Na-inside dependent Ca influx) at either high or low ionic strength. These results suggest that the Ca sites of the Na–Ca exchanger are in a physical environment where they are either not exposed to or not sensitive to surface [Ca].     

New method for visualization of vascular networks in nonperfused fixed tissues

A new method of visualizing the angioarchitecture of tissues has been developed that uses blood components in nonperfused materials. Tissue blocks are fixed in 4% paraformaldehyde and cut with a vibratome into 50-60 micron sections. Endogenous peroxidase in red blood cells is then reduced in the presence of hydrogen peroxide with the resultant oxidation of the chromogen 3,3'-diaminobenzidine (DAB). This generates a dark, highly insoluble reaction product throughout the vascular system. The visualization of vascular components can be further enhanced by exposing the sections to peroxidase-conjugated IgG to increase the background staining of the blood plasma. The technique minimizes preparation artifact and permits the application of morphometric analytical methods, thus allowing parameters such as the volume density of the vascular bed to be quantified.     

New Method for Visualization of Vascular Networks in Nonperfused Fixed Tissues

A new method of visualizing the angioarchitecture of tissues has been developed that uses blood components in nonperfused materials. Tissue blocks are fixed in 4% paraformaldehyde and cut with a vibratome into 50-60 μm sections. Endogenous peroxidase in red blood cells is then reduced in the presence of hydrogen peroxide with the resultant oxidation of the chromogen 3,3'-diaminobenzidine (DAB). This generates a dark, highly insoluble reaction product throughout the vascular system. The visualization of vascular components can be further enhanced by exposing the sections to peroxidase-conjugated IgG to increase the background staining of the blood plasma. The technique minimizes preparation artifact and permits the application of morphometric analytical methods, thus allowing parameters such as the volume density of the vascular bed to be quantified.