Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.     

Smoking behaviour and attitudes among adult Saudi nationals in Riyadh City, Saudi Arabia

Nine massive hemipelvic allografts were used to reconstruct the pelvic ring and the hip articulation after resection of malignant tumors. At follow up of 3 to 10 years, six patients were free of oncologic disease. In the 3 acetabular massive allografts, functional results were close from those standard THR. After resection of hemipelvis and adjacent muscles, patients resume a normal family life (painless hip, poor active motion, walking with a crutch) with a functional result much better than after amputation. Considering these encouraging results in oncologic surgery, we used similar technics for reconstruction of very severe bone loss after iterative failures of THR revisions: some examples are reported at medium follow up.     

Evidence based practice: clinicians' use and attitudes to near patient testing in hospitals

AIM: To survey the use made of laboratory services for urgent tests and clinicians' attitudes to near patient testing. METHODS: A questionnaire was sent to clinicians working in acute hospitals within Trent and North West Thames Regions. RESULTS: 197 replies were received. Most demand came from intensive care units. Overall, clinicians requested a median of six urgent tests a day. Blood glucose and dip stick urine testing were the most commonly performed bedside tests, but 41% of clinicians did not use ward testing. The most frequently cited indication for bedside testing was the need for speed. 85% of clinicians trusted results obtained in their central hospital laboratory, but there was an almost equal division between those who did (34%) and those who did not (38%) trust the results from near patient testing. A slightly larger proportion indicated they would accept responsibility (44%) for results obtained on the ward than would not (35%). Most staff indicated that better transport to the laboratory would remove the need for near patient testing. CONCLUSIONS: Clinicians have demonstrated an apparent need for rapid response testing but there is a strong preference for rapid transport systems and central laboratory analysis rather than bedside testing as a solution to this problem. There is a need to investigate the clinical and cost-effectiveness of near patient testing as a solution to rapid response testing.     

Male factors in infertility--a preliminary report

Findings in 325 consecutive primary seminal analysis done at the Infertility Treatment Centre of IPGMR, are discussed. The clinical profile of the infertile couples was recorded. Semen was examined for quantity, sperm density, motility and morphology. An attempt was also made to correlate past illnesses with the quality of semen. 65 patients (20%) showed azoospermia, while 29 (8.9%) showed sperm count of less than 10 million/ml. History of past illnesses which contribute to azoospermia/oligospermia was obtained in 46% of azoospermia cases compared to only 10% in subjects showing sperm count of over 10 million/ml. Commonest association between azoospermia and past illness was found in the case of small-pox, where out of 31 subjects with past history of small-pox, 15 showed complete azoospermia, one showed sperm density of less then 10 million/ml.     

Structure and architecture of the bacterial virus fd. An infrared linear dichroism study

Oriented gels of intact bacterial virus fd have been invetigated by infrared linear dichroism. Infrared absorption band maxima and dichroism indicate an alpha-helix content of the major coat protein of 95-100%. The alpha-helical rods of the coat protein are aligned parallel to the long axis of the virion with an inclination roughly estimated to approximately 37 degree. The presence of DNA infrared bands at 968, 885, 830 and 799 cm-1, the absence of a band at 860 cm-1 and the perpendicular polarization of the symmetric PO-2 stretching vibration at 1085 cm-1 are all indicative of a B-type backbone conformation in the single-stranded DNA. We find no evidence for specific interaction between aromatic side groups (phenylalanine, tyrosine) and the DNA bases. Our results independently confirm most features of the model of Marvin and co-workers [2,15 ] based on low-resolution X-ray diffraction studies. However, our findings contradict their suggestion of an A-type DNA in the bacterial virus fd. Two results are consistent with rigid and stable order in the virus. First, over a 4-day period, 65% of the peptide hydrogens remain unexchanged with deuterium. Second, changes in the relative humidity of the sample do not result in any shifts in the DNA spectrum that are characteristic of free DNA.     

A simple, general procedure for purifying restriction endonucleases

A simple, general method for purifying restriction endonucleases is described. The method employs precipitation of nucleic acids from crude extracts with polyethyleneimine followed by affinity chromatography on columns of heparin covalently linked to agarose. Most of the sixteen enzymes tested could be purified to a degree sufficient for DNA sequencing work by this method sometimes supplemented by at most one step of ion exchange chromatography.     

Structural characteristics of the mediodorsal nucleus of the cat thalamus

By means of the light and electron microscopy, construction and architectonics of neurons in the cat thalamus medio-dorsal nucleus are described. Differences in the neuronal body dimentions, their form, number of deferent dendrites and character of axonal branching made it possible to define 3 types of neurons. Applying the method based on retrograde axonal transport of exogenous horse-radish peroxidase, it was demonstrated that neurons of type II, contrary to the existing opinion, send their axons into the prefrontal cortex and, hence, are associative-projective. Ultrastructural difference of axonal terminals, differences in the form and size of synaptic vesicles made it possible to reveal 5 types of presynapses.