Observation of magnet-induced star-like radiation of a plasma created from cancer cells in a laser trap.

We present a new phenomenon resulting from the interaction of magnetic beads with cancer cells in a laser trap formed on a slide containing a depression 16.5 mm in diameter and 0.78 mm of maximum depth. This phenomenon includes the apparent formation and expansion of a dark bubble that attracts and incinerates surrounding matter when it explodes, which leads to a plasma emitting intense radiation that has the appearance of a star on a microscopic scale. We have observed the star-like phenomenon for more than 4 years, and the intensity depends on the laser's power. Measuring the laser power of the dark bubble shows the entrapment of electromagnetic energy as it expands.

Transcutaneous oximetry in clinical practice: consensus statements from an expert panel based on evidence.

Transcutaneous oximetry (PtcO2) is finding increasing application as a diagnostic tool to assess the peri-wound oxygen tension of wounds, ulcers, and skin flaps. It must be remembered that PtcO2 measures the oxygen partial pressure in adjacent areas of a wound and does not represent the actual partial pressure of oxygen within the wound, which is extremely difficult to perform. To provide clinical practice guidelines, an expert panel was convened with participants drawn from the transcutaneous oximetry workshop held on June 13, 2007, in Maui, Hawaii. Important consensus statements were (a) tissue hypoxia is defined as a PtcO2 <40 mm Hg; (b) in patients without vascular disease, PtcO2 values on the extremity increase to a value >100 mm Hg when breathing 100% oxygen under normobaric pressures; (c) patients with critical limb ischemia (ankle systolic pressure of < or =50 mm Hg or toe systolic pressure of < or =30 mm Hg) breathing air will usually have a PtcO2 <30 mm Hg; (d) low PtcO2 values obtained while breathing normobaric air can be caused by a diffusion barrier; (e) a PtcO2 <40 mm Hg obtained while breathing normobaric air is associated with a reduced likelihood of amputation healing; (f) if the baseline PtcO2 increases <10 mm Hg while breathing 100% normobaric oxygen, this is at least 68% accurate in predicting failure of healing post-amputation; (g) an increase in PtcO2 to >40 mm Hg during normobaric air breathing after revascularization is usually associated with subsequent healing, although the increase in PtcO2 may be delayed; (h) PtcO2 obtained while breathing normobaric air can assist in identifying which patients will not heal spontaneously.

Determination of ideal PtcO2 measurement time in evaluation of hypoxic wound patients.

OBJECTIVE: Evaluation of ideal time for baseline PtcO2 readings in air, elevation test, and oxygen challenge during evaluation of hypoxic wound patients. DESIGN: Retrospective analysis. IRB APPROVAL: Western IRB deemed this study exempt from requiring IRB approval. PATIENTS: 202 patients with lower extremity wounds. METHOD: Patients had PtcO2 measurements using 6 electrodes positioned in 3 paired locations along the limb (above the knee: AK; below the knee: BK; and foot). Measurements were made from each electrode at 7 different time-event occasions: position of limb (supine or elevated), type of breathing gas (sea level air or oxygen), and time of measurement. A total of 8,484 measurements were analyzed by first examining each electrode's data, and then pooling the data for each location pair. MAIN RESULTS: PtcO2 readings for air (10 minutes) were less than air at 20 minutes. Maximal readings were close to the 20-minute mark for AK and BK measurements, and closer to 30 minutes for the foot. Elevation test at 3 versus 5 minutes showed a continuing decline in PtcO2 values. Oxygen challenge readings at 5 and 10 minutes were significantly different: the latter always larger than the former. CONCLUSION: Ideal times for baseline readings, leg elevation test, and oxygen challenge test are at least 20, 5, and 10 minutes, respectively.

Borg proteins control septin organization and are negatively regulated by Cdc42.

The Cdc42 GTPase binds to numerous effector proteins that control cell polarity, cytoskeletal remodelling and vesicle transport. In many cases the signalling pathways downstream of these effectors are not known. Here we show that the Cdc42 effectors Borg1 to Borg3 bind to septin GTPases. Endogenous septin Cdc10 and Borg3 proteins can be immunoprecipitated together by an anti-Borg3 antibody. The ectopic expression of Borgs disrupts normal septin organization. Cdc42 negatively regulates this effect and inhibits the binding of Borg3 to septins. Borgs are therefore the first known regulators of mammalian septin organization and provide an unexpected link between the septin and Cdc42 GTPases.

Preparation and crystal structure of the recombinant alpha(1)/alpha(2) catalytic heterodimer of bovine brain platelet-activating factor acetylhydrolase Ib.

The intracellular form of mammalian platelet activating factor acetylhydrolase found in brain (PAF-AH Ib) is thought to play a critical role in control in neuronal migration during cortex development. This oligomeric complex consists of a homodimer of the 45 kDa (beta) LIS1 protein, the product of the causative gene for type I lissencephaly, and, depending on the developmental stage and species, one of three possible pairs of two homologous approximately 26 kDa alpha-subunits, which harbor all of the catalytic activity. The exact composition of this complex depends on the expression patterns of the alpha(1) and alpha(2) genes, exhibiting tissue specificity and developmental control. All three possible dimers (alpha(1)/alpha(1), alpha(1)/alpha(2) and alpha(2)/alpha(2)) were identified in tissues. The alpha(1)/alpha(2) heterodimer is thought to play an important role in fetal brain. The structure of the alpha(1)/alpha(1) homodimer was solved earlier in our laboratory at 1.7 A. We report here the preparation of recombinant alpha(1)/alpha(2) heterodimers using a specially constructed bi-cistronic expression vector. The approach may be useful in studies of other systems where pure heterodimers of recombinant proteins are required. The alpha(1)/alpha(2) dimer has been crystallized and its structure was solved at 2.1 A resolution by molecular replacement. These results set the stage for a detailed characterization of the PAF-AH Ib complex.

Expression, purification, and crystallization of the RGS-like domain from the Rho nucleotide exchange factor, PDZ-RhoGEF, using the surface entropy reduction approach.

Lsc-homology domains are found in several eukaryotic nucleotide exchange factors which act on Rho-family GTPases. They show limited amino acid sequence similarity to RGS proteins, which down-regulate the cellular signaling by the alpha-subunits of trimeric G-proteins and have been shown to interact with Galpha12 and Galpha13. It is believed that the RGS-like (RGSL) domain constitutes the functional link between G-protein-coupled receptors and cytosolic Rho-GTPases. We report here the expression, purification, and crystallization of the RGSL domain from the PDZ-RhoGEF. To obtain X-ray-grade crystals we have used the recently proposed approach of crystallization by mutational surface entropy reduction, in which selected Lys --> Ala, Glu --> Ala, and/or combined point mutations are introduced into the target protein to reduce the cumulative conformational entropy of surface residues. Of the five mutants that were designed and prepared, the second one tried (K463A, E465A, E466A) yielded crystals suitable for further analysis and diffracted X-rays to 2.8 A resolution on a home source. The crystals exhibit hexagonal symmetry, space group P6(1) 22 or P6(5) 22, with unit cell parameters a = b = 63.1 A, c = 202.1 A, and contain one molecule in the asymmetric unit.

Protein crystallization by rational mutagenesis of surface residues: Lys to Ala mutations promote crystallization of RhoGDI.

Crystallization is a unique process that occurs at the expense of entropy, including the conformational entropy of surface residues, which become ordered in crystal lattices during formation of crystal contacts. It could therefore be argued that epitopes free of amino acids with high conformational entropy are more thermodynamically favorable for crystal formation. For a protein recalcitrant to crystallization, mutation of such surface amino acids to residues with no conformational entropy might lead to enhancement of crystallization. This paper reports the results of experiments with an important cytosolic regulator of GTPases, human RhoGDI, in which lysine residues were systematically mutated to alanines. Single and multiple mutations were introduced into two different variants of RhoGDI, NDelta23 and NDelta66, in which the first 23 and 66 residues, respectively, were removed by recombinant methods. In total, 13 single and multiple mutants were prepared and assessed for crystallization and all were shown to crystallize using the Hampton Research Crystal Screens I and II, in contrast to wild-type NDelta23 and NDelta66 RhoGDI which did not crystallize. Four crystal structures were solved (the triple mutants NDelta23:K135,138,141A and NDelta66:K135,138,141A, and two single mutants NDelta66:K113A and NDelta66:K141A) and in three cases the crystal contacts of the new lattices were found precisely at the sites of mutations. These results support the notion that it is, in principle, possible to rationally design mutations which systematically enhance proteins' ability to crystallize.

Structure of the BH domain from graf and its implications for Rho GTPase recognition.

Cellular signaling by small G-proteins is down-regulated by GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis. The GTPase regulator associated with focal adhesion kinase (Graf) exhibits GAP activity toward the RhoA and Cdc42 GTPases, but is only weakly active toward the closely related Rac1. We determined the crystal structure of a 231-residue fragment of Graf (GrafGAP), a domain containing the GAP activity, at 2.4-A resolution. The structure clarifies the boundaries of the functional domain and yields insight to the mechanism of substrate recognition. Modeling its interaction with substrate suggested that a favorable interaction with Glu-95 of Cdc42 (Glu-97 of RhoA) would be absent with the corresponding Ala-95 of Rac1. Indeed, GrafGAP activity is diminished approximately 40-fold toward a Cdc42 E95A mutant, whereas a approximately 10-fold increase is observed for a Rac1 A95E mutant. The GrafGAP epitope that apparently interacts with Glu-95(Glu-97) contains Asn-225, which was recently found mutated in some myeloid leukemia patients. We conclude that position 95 of the GTPase is an important determinant for GrafGAP specificity in cellular function and tumor suppression.

Hyperbaric oxygen in the treatment of delayed radiation injuries of the extremities.

Hyperbaric oxygen (HBO2) is used as an adjunct in the treatment of radiation injury at many sites, including the mandible, larynx, chest wall, bladder, and rectum. In these disorders, HBO2 is effective in stimulating neovascularization and reducing fibrosis. No previous publications report the application of HBO2 to radiation injuries of the extremities. From 1979 until 1997, 17 patients were treated at the Southwest Texas Methodist and Nix Hospitals for nonhealing necrotic wounds of the extremities within previously irradiated fields. All but one wound involved a lower extremity. Most of the patients had been irradiated for soft tissue sarcomas or skin cancers. The rest were irradiated for a variety of malignancies. HBO2 was delivered in a multiplace chamber at 2.4 atm abs daily for 90 min of 100% oxygen at pressure. This report is a retrospective, uncontrolled review of these patients. Eleven patients (65%) healed completely whereas five (29%) failed to heal and one (6%) was lost to follow-up. Three (60%) of those who failed were found to have local or distant recurrence of their tumor early in their course of hyperbaric treatment and were discontinued from therapy at that time. When last seen in the clinic, the wound of the patient who was lost to follow-up was improved but not completely healed. Four of those who failed (including the two with local tumor recurrence) required amputation. If we exclude those with active cancer and the patient lost to follow-up, the success rate was 11 of 13 or 85%. HBO2 was applied successfully with complete wound healing and the avoidance of amputation in a majority of these patients. The consequences of failure in patients suffering from radiation necrosis of the extremities (some complicated by the presence of tumor) are significant, with 80% of the five failures requiring amputation. In radiation injuries of the extremities as in delayed radiation injury at other sites, HBO2 is a useful adjunct and should be part of the overall management.

Homologs of the alpha- and beta-subunits of mammalian brain platelet-activating factor acetylhydrolase Ib in the Drosophila melanogaster genome.

The mammalian intracellular brain platelet-activating factor acetylhydrolase, implicated in the development of cerebral cortex, is a member of the phospholipase A2 superfamily. It is made up of a homodimer of the 45 kDa LIS1 protein (a product of the causative gene for type I lissencephaly) and a pair of homologous 26-kDa alpha-subunits which account for all the catalytic activity. LIS1 is hypothesized to regulate nuclear movement in migrating neurons through interactions with the cytoskeleton, while the alpha-subunits, whose structure is known, contain a trypsin-like triad within the framework of a unique tertiary fold. The physiological significance of the association of the two types of subunits is not known. In an effort to better understand the function of the complex we turned to genomic data mining in search of related proteins in lower eukaryotes. We found that the Drosophila melanogaster genome contains homologs of both alpha- and beta-subunits, and we cloned both genes. The alpha-subunit homolog has been overexpressed, purified and crystallized. It lacks two of the three active-site residues and, consequently, is catalytically inactive against PAF-AH (Ib) substrates. Our study shows that the beta-subunit homolog is highly conserved from Drosophila to mammals and is able to interact with the mammalian alpha-subunits but is unable to interact with the Drosophila alpha-subunit. Proteins 2000;39:1-8.

The functional implications of the dimerization of the catalytic subunits of the mammalian brain platelet-activating factor acetylhydrolase (Ib).

The mammalian brain contains significant amounts of the cytosolic isoform Ib of the platelet-activating factor acetylhydrolase (PAF-AH), a unique type of PLA2. This oligomeric protein complex contains three types of subunits: two homologous (63% identity) 26 kDa catalytic subunits (alpha(1) and alpha(2)) which harbor all the PAF-AH activity, and the 45 kDa beta-subunit (LIS1), a product of the causal gene for Miller-Dieker lissencephaly. During fetal development, the preferentially expressed alpha(1)-subunit forms a homodimer, which binds to a homodimer of LIS1, whereas in adult organisms alpha(1)/alpha(2) and alpha(2)/alpha(2) dimers, also bound to dimeric LIS1, are the prevailing species. The consequences of this "switching" are not understood, but appear to be of physiological significance. The alpha(1)- and alpha(2)-subunits readily associate with very high affinity to form homodimers. The nature of the interface has been elucidated by the 1.7 A resolution crystal structure of the alpha(1)/alpha(1) homodimer (Ho et al., 1997). Here, we examined the functional consequences of the dimerization in both types of alpha-subunits. We obtained monomeric protein in the presence of high concentrations (>50 mM) of Ca2+ ions, and we show that it is catalytically inactive and less stable than the wild type. We further show that Arg29 and Arg22 in one monomer contribute to the catalytic competence of the active site across the dimer interface, and complement the catalytic triad of Ser47, Asp192 and His195, in the second monomer. These results indicate that the brain PAF-acetylhydrolase is a unique PLA2 in which dimerization is essential for both stability and catalytic activity.

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.

Expression, purification and crystallization of a BH domain from the GTPase regulatory protein associated with focal adhesion kinase.

Signaling by small GTPases is down-regulated by GTPase activating proteins (GAPs) which enhance the rate of GTP hydrolysis. The activity of GAPs specific for Rho GTPases resides in the BH domain, many homologues of which are found in any mammalian genome. One of them was identified in the GTPase regulator associated with focal-adhesion kinase (GRAF). It shares approximately 20% sequence identity with p50RhoGAP. This GAP activates RhoA and Cdc42Hs, but not Rac. In order to dissect the molecular basis of this specificity, a 231-residue-long fragment corresponding to the BH domain of GRAF has been expressed, purified and crystallized. Trigonal crystals, of space group P3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 A were grown from solutions of PEG 6000. Data to 2.15 A were collected from a flash-frozen sample on an R-AXIS IV imaging-plate detector mounted on a rotating anode X-ray generator.

Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase.

Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication. We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence. The correct sequence, inferred from the experimentally phased electron density map, revealed that BFAE is a homolog of the mammalian hormone sensitive lipase (HSL). It is a canonical alpha/beta hydrolase with two insertions forming the substrate binding pocket. The enzyme contains a lipase-like catalytic triad, Ser 202, Asp 308 and His 338, consistent with mutational studies that implicate the homologous Ser 424, Asp 693 and His 723 in the catalytic triad in human HSL.

Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors.

We describe the construction of expression vectors based on three of the most frequently used gene fusion affinity tags [glutathione S-transferase (GST), maltose binding protein (MBP), and the His6 peptide]. The polylinkers of pGEX4T1, pMal-c2, and a pET vector were replaced with the polylinker isolated from the baculovirus expression plasmid pFastBac. Once appropriate restriction sites have been introduced into a gene, it can be fused to all three affinity tags with little effort, allowing expression-screening experiments to be performed efficiently. We discuss the development and use of these vectors with respect to overcoming purification problems encountered for the RhoA GDP/GTP nucleotide dissociation inhibitor (RhoGDI) and their advantages over commercially available expression vectors.

Promoter analysis of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa.

Analysis of the promoter region of the acetate-induced isocitrate lyase gene (acu-3) of Neurospora crassa was undertaken. A series of deletions in the 5' non-transcribed region were constructed and the effects of these mutations on the enzyme levels following growth on sucrose and transfer to acetate were measured. Sequences within the region -603 to -271 relative to the transcription start site appear essential for transcription. The region -950 to -1278 is required for sucrose repression, which is consistent with previous protein/DNA gel retardation results of protein extracts from N. crassa cultured on sucrose. Protein extracts from acetate-induced mycelia identify alternative promoter regions apparently involved in acetate-induced gene transcription.

Measuring tissue oxygen tension: a review.

Because of technological advances in tissue oximetry, clinicians and scientists have a better understanding of the role of oxygen in wound healing. In wound care and hyperbaric medicine applications, an oximeter is principally used with vascular assessment to help determine amputation level and to estimate healing potential. With the current emphasis on cost savings in the managed care setting, transcutaneous oximetry (PtcO2) has gained importance as a tool for predicting potential candidates for hyperbaric oxygen (HBO2) therapy. It is used to identify the presence of hypoxia in wounded tissue, to predict the responders to hyperoxia and in some instances to determine when HBO2 treatment is complete. This literature review describes the principal current methods for measuring tissue O2 and the values obtained in normal and wounded tissue under both normobaric and hyperbaric conditions. The review includes the Jefferson C. Davis Wound Care and Hyperbaric Medicine Center protocol for PtcO2 assessment of potential HBO2 candidates and suggestions for obtaining reproducible PtcO2 data.

Hyperbaric and hypobaric chamber fires: a 73-year analysis.

Fire can be catastrophic in the confined space of a hyperbaric chamber. From 1923 to 1996, 77 human fatalities occurred in 35 hyperbaric chamber fires, three human fatalities in a pressurized Apollo Command Module, and two human fatalities in three hypobaric chamber fires reported in Asia, Europe, and North America. Two fires occurred in diving bells, eight occurred in recompression (or decompression) chambers, and 25 occurred in clinical hyperbaric chambers. No fire fatalities were reported in the clinical hyperbaric chambers of North America. Chamber fires before 1980 were principally caused by electrical ignition. Since 1980, chamber fires have been primarily caused by prohibited sources of ignition that an occupant carried inside the chamber. Each fatal chamber fire has occurred in an enriched oxygen atmosphere (> 28% oxygen) and in the presence of abundant burnable material. Chambers pressurized with air (< 23.5% oxygen) had the only survivors. Information in this report was obtained from the literature and from the Undersea and Hyperbaric Medical Society's Chamber Experience and Mishap Database. This epidemiologic review focuses on information learned from critical analyses of chamber fires and how it can be applied to safe operation of hypobaric and hyperbaric chambers.