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PubMed2005

Laterally transferred elements and high pressure adaptation in Photobacterium profundum strains.

BACKGROUND: Oceans cover approximately 70% of the Earth's surface with an average depth of 3800 m and a pressure of 38 MPa, thus a large part of the biosphere is occupied by high pressure environments. Piezophilic (pressure-loving) organisms are adapted to deep-sea life and grow optimally at pressures higher than 0.1 MPa. To better understand high pressure adaptation from a genomic point of view three different Photobacterium profundum strains were compared. Using the sequenced piezophile P. profundum strain SS9 as a reference, microarray technology was used to identify the genomic regions missing in two other strains: a pressure adapted strain (named DSJ4) and a pressure-sensitive strain (named 3TCK). Finally, the transcriptome of SS9 grown under different pressure (28 MPa; 45 MPa) and temperature (4 degrees C; 16 degrees C) conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool. RESULTS: These studies indicated the presence of a large flexible gene pool in SS9 characterized by various horizontally acquired elements. This was verified by extensive analysis of GC content, codon usage and genomic signature of the SS9 genome. 171 open reading frames (ORFs) were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. Among these genes, six were found to also be up-regulated by high pressure. CONCLUSION: These data provide information on horizontal gene flow in the deep sea, provide additional details of P. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth.

Atmospheric Pressure↗
PubMed2006

The influence of breathing motion on intensity modulated radiotherapy in the step-and-shoot technique: phantom measurements for irradiation of superficial target volumes.

For intensity modulated radiotherapy (IMRT) of deep-seated tumours, dosimetric variations of the original static dose profiles due to breathing motion can be primarily considered as blurring effects known from conventional radiotherapy. The purpose of this dosimetric study was to clarify whether these results are transferable to superficial targets and to quantify the additional effect of fractionation. A solid polystyrene phantom and an anthropomorphic phantom were used for film and ion chamber dose measurements. The phantoms were installed on an electric driven device and moved with a frequency of 6 or 12 cycles per minute and an amplitude of 4 mm or 10 mm. A split beam geometry of two adjacent asymmetric fields and an IMRT treatment plan with 12 fields for irradiation of the breast were investigated. For the split beam geometry the dose modifications due to unintended superposition of partial fields were reduced by fractionation and completely smoothed out after 20 fractions. IMRT applied to the moving phantom led to a more homogeneous dose distribution compared to the static phantom. The standard deviation of the target dose which is a measure of the dose homogeneity was 10.3 cGy for the static phantom and 7.7 cGy for a 10 mm amplitude. The absolute dose values, measured with ionization chambers, remained unaffected. Irradiation of superficial targets by IMRT in the step-and-shoot technique did not result in unexpected dose perturbations due to breathing motion. We conclude that regular breathing motion does not jeopardize IMRT of superficial target volumes.

Breast↗
PubMed2004

Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.

The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.

Amino Acid Sequence↗
PubMed2004

Crystal structures of cobalamin-independent methionine synthase complexed with zinc, homocysteine, and methyltetrahydrofolate.

Cobalamin-independent methionine synthase (MetE) catalyzes the synthesis of methionine by a direct transfer of the methyl group of N5-methyltetrahydrofolate (CH3-H2PteGlun) to the sulfur atom of homocysteine (Hcy). We report here the first crystal structure of this metalloenzyme under different forms, free or complexed with the Hcy and folate substrates. The Arabidopsis thaliana MetE (AtMetE) crystals reveal a monomeric structure built by two (betaalpha)8 barrels making a deep groove at their interface. The active site is located at the surface of the C-terminal domain, facing the large interdomain cleft. Inside the active site, His647, Cys649, and Cys733 are involved in zinc coordination, whereas Asp605, Ile437, and Ser439 interact with Hcy. Opposite the zinc/Hcy binding site, a cationic loop (residues 507-529) belonging to the C-terminal domain anchors the first glutamyl residue of CH3-H4PteGlu5. The pterin moiety of CH3-H4PteGlu5 is stacked with Trp567, enabling the N5-methyl group to protrude in the direction of the zinc atom. These data suggest a structural role of the N-terminal domain of AtMetE in the stabilization of loop 507-529 and in the interaction with the poly-glutamate chain of CH3-H4PteGlun. Comparison of AtMetE structures reveals that the addition of Hcy does not lead to a direct coordination of the sulfur atom with zinc but to a reorganization of the zinc binding site with a stronger coordination to Cys649, Cys733, and a water molecule.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2003

Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form.

The E. coli nitroreductase enzyme (NTR) has been widely used in suicide gene therapy (GDEPT and ADEPT) applications as a activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class. NTR has been previously shown to be a homodimeric enzyme with two active sites. We present here the crystal structures of the reduced form of NTR and its complexes with the inhibitor dicoumarol and three dinitrobenzamide prodrugs. Comparison of the structures of the oxidized and reduced forms of the native enzyme shows that the principal structural changes occur in the FMN cofactor and indicate that the enzyme itself is a relatively rigid structure that primarily provides a rigid structural framework on which hydride transfer occurs. The aziridinyldinitrobenzamide prodrug CB 1954 binds in nonidentical ways in both of the two active sites of the homodimeric enzyme, employing both hydrophobic and (in active site B) a direct H-bond contact to the side chain of Lys14. In active site A the 2-nitro group stacks above the FMN, and in active site B the 4-nitro group does, explaining why reduction of either nitro group is observed. In contrast, the larger mustard group of the dinitrobenzamide mustard compound SN 23862 forces the prodrug to bind at both active sites with only the 2-nitro group able to participate in hydride transfer from the FMN, explaining why only the 2-hydroxylamine reduction product is observed. In each site, the nitro groups of the prodrug make direct H-bond contacts with the enzyme; in active Site A the 2-nitro to Ser40 and the 4-nitro to Asn71, while in active Site B the 2-nitro contacts the main chain nitrogen of Thr41 and the 4-nitro group the Lys14 side chain. The related amide-substituted mustard SN 27217 binds in a broadly similar fashion, but with the larger amide group substituent able to reach and contact the side chain of Arg107, further restricting the prodrug conformations in the binding site. The inhibitor dicoumarol appears to bind primarily by pi-stacking interactions and hydrophobic contacts, with no conformational changes in the enzyme. One of the hydroxycoumarin subunits stacks above the plane of the FMN via pi-overlap with the isoalloxazine ring, penetrating deep into the groove, with the other less well-defined. These studies suggest guidelines for further prodrug design. Steric bulk (e.g., mustard rather than aziridine) on the ring can limit the possible binding orientations, and the reducible nitro group must be located para to the mustard. Substitution on the carboxamide side chain still allows the prodrugs to bind, but also limits their orientation in the binding site. Finally, modulating substrate specificity by alteration of the structure of the enzyme rather than the prodrug might usefully focus on modifying the Phe124 residue and those surrounding it.

Aniline Mustard↗
PubMed2004

Investigation of TRAM flap oxygenation and perfusion by near-infrared reflection spectroscopy and color-coded duplex sonography.

Near-infrared reflection spectroscopy, used experimentally for investigation of tissue hemoglobin content and oxygenation in various flaps, was tested in the pedicled transverse rectus abdominis musculocutaneous (TRAM) flap, chosen as a simple clinical model because of its well-known vascular anatomy and clinical relevance. The study intended to answer the following questions: Does the near-infrared reflection spectroscopy system used in this study measure tissue hemoglobin content and oxygenation in the superficial skin layers only, as proposed by the manufacturer? Is near-infrared reflection spectroscopy able to detect differences of tissue hemoglobin content and oxygenation in distinct zones of the TRAM flap skin before, early, and late after surgery? Does tissue hemoglobin content and oxygenation correspond to blood flow in the supplying superior epigastric artery and to clinical signs of TRAM flap perfusion and viability? In 11 patients, tissue hemoglobin content and oxygenation in the lower abdomen/TRAM flap, mastectomy skin flap, and contralateral breast were measured by a new near-infrared reflection spectroscopy system preoperatively, early postoperatively, and late postoperatively. Simultaneously, systolic peak flow in the ipsilateral superior epigastric artery was obtained by color-coded duplex sonography. Routine clinical monitoring was performed throughout the early postoperative period. Tissue hemoglobin content and oxygenation in the lower abdomen, mastectomy skin flap, and contralateral breast were similar before surgery but varied considerably between different patients. There were no significant differences among preoperative, early postoperative, and late postoperative values of tissue hemoglobin content and oxygenation in the mastectomy skin flap and contralateral breast. However, near-infrared reflection spectroscopy measurements of the TRAM flap revealed significant differences between preoperative and early postoperative values of tissue hemoglobin content and oxygenation and among zones I, II, and III early after surgery. Tissue hemoglobin content in the TRAM flap skin increased and oxygenation decreased early after surgery. Near-infrared reflection spectroscopy values corresponded to clinical signs of venous congestion predominantly in zone III. Late postoperative return of hemoglobin content and oxygenation in the TRAM flap toward preoperative values can be attributed to improved venous return by reversed flow across regurgitant valves and development of collateral circulation. Finally, there was a significant increase of systolic peak flow in the ipsilateral superior epigastric artery early after surgery. This could be related to the opening of small-caliber choke arteries between the superior and deep inferior epigastric arteries following ligation of the dominant deep inferior epigastric artery and TRAM flap transfer to the chest. Systolic peak flow returned to preoperative values late after surgery. The near-infrared reflection spectroscopy system used in this study appeared to measure hemoglobin content and oxygenation in the superficial skin layers only. Near-infrared reflection spectroscopy was able to detect differences of tissue hemoglobin content and oxygenation in the TRAM flap between preoperative and postoperative measurements and between distinct zones of the TRAM flap early postoperatively. Postoperative changes in near-infrared reflection spectroscopy values corresponded to clinical observations and blood flow in the superior epigastric artery measured by color-coded duplex sonography. Further experience is needed before near-infrared reflection spectroscopy can be advocated for routine clinical flap monitoring.

Adult↗
PubMed2005

A new type of sulfite reductase, a novel coenzyme F420-dependent enzyme, from the methanarchaeon Methanocaldococcus jannaschii.

Methanocaldococcus jannaschii is a hypertheromphilic, strictly hydrogenotrophic, methanogenic archaeon of ancient lineage isolated from a deep-sea hydrothermal vent. It requires sulfide for growth. Sulfite is inhibitory to the methanogens. Yet, we observed that M. jannaschii grows and produces methane with sulfite as the sole sulfur source. We found that in this organism sulfite induces a novel, highly active, coenzyme F(420)-dependent sulfite reductase (Fsr) with a cell extract specific activity of 0.57 mumol sulfite reduced min(-1) mg(-1) protein. The cellular level of Fsr protein is comparable to that of methyl-coenzyme M reductase, an enzyme essential for methanogenesis and a possible target for sulfite. Purified Fsr reduces sulfite to sulfide using reduced F(420) (H(2)F(420)) as the electron source (K(m): sulfite, 12 microm; H(2)F(420), 21 microm). Therefore, Fsr provides M. jannaschii an anabolic ability and protection from sulfite toxicity. The N-terminal half of the 70-kDa Fsr polypeptide represents a H(2)F(420) dehydrogenase and the C-terminal half a dissimilatory-type siroheme sulfite reductase, and Fsr catalyzes the corresponding partial reactions. Previously described sulfite reductases use nicotinamides and cytochromes as electron carriers. Therefore, this is the first report of a coenzyme F(420)-dependent sulfite reductase. Fsr homologs were found only in Methanopyrus kandleri and Methanothermobacter thermautotrophicus, two strictly hydrogenotrophic thermophilic methanogens. fsr is the likely ancestor of H(2)F(420) dehydrogenases, which serve as electron input units for membrane-based energy transduction systems of certain late evolving archaea, and dissimilatory sulfite reductases of bacteria and archaea. fsr could also have arisen from lateral gene transfer and gene fusion events.

Amino Acid Sequence↗
PubMed1999

Comparison of four magnetic resonance methods for mapping small temperature changes.

Non-invasive detection of small temperature changes (< 1 degree C) is pivotal to the further advance of regional hyperthermia as a treatment modality for deep-seated tumours. Magnetic resonance (MR) thermography methods are considered to be a promising approach. Four methods exploiting temperature-dependent parameters were evaluated in phantom experiments. The investigated temperature indicators were spin-lattice relaxation time T1, diffusion coefficient D, shift of water proton resonance frequency (water PRF) and resonance frequency shift of the methoxy group of the praseodymium complex (Pr probe). The respective pulse sequences employed to detect temperature-dependent signal changes were the multiple readout single inversion recovery (T One by Multiple Read Out Pulses; TOMROP), the pulsed gradient spin echo (PGSE), the fast low-angle shot (FLASH) with phase difference reconstruction, and the classical chemical shift imaging (CSI). Applying these sequences, experiments were performed in two separate and consecutive steps. In the first step, calibration curves were recorded for all four methods. In the second step, applying these calibration data, maps of temperature changes were generated and verified. With the equal total acquisition time of approximately 4 min for all four methods, the uncertainties of temperature changes derived from the calibration curves were less than 1 degree C (Pr probe 0.11 degrees C, water PRF 0.22 degrees C, D 0.48 degrees C and T1 0.93 degrees C). The corresponding maps of temperature changes exhibited slightly higher errors but still in the range or less than 1 degree C (0.97 degrees C, 0.41 degrees C, 0.70 degrees C, 1.06 degrees C respectively). The calibration results indicate the Pr probe method to be most sensitive and accurate. However, this advantage could only be partially transferred to the thermographic maps because of the coarse 16 x 16 matrix of the classical CSI sequence. Therefore, at present the water PRF method appears to be most suitable for MR monitoring of small temperature changes during hyperthermia treatment.

Body Temperature↗
PubMed2004

[Experimental and clinical study on functional restoration of the penis with large partial defect].

OBJECTIVE: To investigate the effective restoration method in penile partial defect. METHODS: To measure the thickness of suspensory ligaments on cadavers; To find the relationship between inferior pubic ramus and corpora cavernosa crus through corpora cavernosa angiography and ultrasonic scanning. The procedure of restoration was based on the use of dissection of the suspensory ligaments and part of crus, and the local fat flap had been transferred to fill the front space of symphysis ossium pubi. Various flaps were designed as coverage material. Penis residual stump was advanced to anterior portion of the newly restoration penile body as "glans"; To take full layer skin of penis from post-operating patients to observe neurofibril regeneration by light microscopy, electron microscopy and scanning electron microscopy; To determine sensation recovery by physical examination; To check erectile function with NEVA System. RESULTS: The whole suspensory ligaments thickness was 7.66 cm in average on 30 cadavers (60 sides). The crus were attached to bilateral inferior pubic ramus respectively, and distal reached the middle of inferior pubic ramus by corpora cavernosa angiography. In 14 health men by ultrasonic scanning, 6.60 cm in average from root of external penis to location of deep artery penis entered corpora cavernosa. Forty patients had been operated with the above methods. In the cases, the length of the penis varied from 0.5-4.0 cm in the flaccid, 2.0-6.0 cm in erect state before operation to 5.0-8.5 cm in the flaccid, 7.0-12.5 cm in erect state after operation. Twenty-eight cases had been followed up for more than one year, and 23 patients married with acceptable sexual life, eighteen men had successfully produced their next generations. The regenerative neurofibril could be seen under light microscope, immunohistochemistry marker, scanning and transmission electron microscope. The penis presented normal pain and touch feeling in the post-operating cases. The results showed that there was similar erectile ability of a normal male in the follow-up patients. CONCLUSION: With these basic and clinical researches, the restoration of penis tends to have a better appearance and function. It's a more optimal method compared with conventional procedures. The results have also provided valuable data for further study in related fields.

Adult↗
PubMed1992

Needlestick injury: blood, mononuclear cells, and acquired immunodeficiency syndrome.

INTRODUCTION: Because transmission of HIV to health care workers after needlestick injury has occurred mainly a result of deep insertion of large gauge needles, blood and viable mononuclear cells transferred after needlestick injury were measured. METHODS: Needles of 20 to 27 gauge were filled with HIV-1 seropositive blood and inserted through extracorporeal human skin or parafilm covering physiologic saline solution modified Drabkin's solution, or culture medium, or inserted directly into one of these fluids, to a depth of one third of the needle length (0.5 inch) for 1 second. Volume of blood transferred was measured by both modified Drabkin's method and by chromium 51 labeling of red blood cells. Transfer of viable mononuclear cells was measured by growth in culture medium containing autologous feeder cells. RESULTS: The volume of blood transferred from a needle passed through skin varied from 312 +/- 69 nl from a 20-gauge needle to 14 +/- 4 nl from a 27-gauge needle, as measured by modified Drabkin's technique, and from 404 +/- 80 nl to 12 +/- 3.1 nl, as measured by chromium 51 labeling of red blood cells. The volume of blood transferred from a needle passed through parafilm was twice that transferred through skin. The volume of blood transferred through skin was 40% that transferred directly into fluid not covered by any barrier; blood transferred through parafilm was 80% of that transferred directly. When needles containing blood were inserted into culture medium for 1 second in the absence of a barrier, at least one viable mononuclear cell was almost always transferred to fluid from all gauges of needle tested. Insertion of needles through skin prevented transfer of all viable mononuclear cells from only 3% to 5% of 20- to 23-gauge needles, and from 12% to 32% of 26- and 27-gauge needles. Parafilm was an even less effective barrier than skin. Insertion of needles through parafilm completely prevented transfer of viable mononuclear cells from no 20- to 23-gauge needles and from only 5% to 10% of 26- and 27-gauge needles. CONCLUSION: The volume of blood transferred after needle insertion through skin for 1 second varied with the gauge of the needle and was 30-fold higher from a 20-gauge than from a 27-gauge needle. Variable mononuclear cells were transmitted after insertion through skin from more than 95% of 20- to 23-gauge needles and from 68% to 88% of 26- and 27-gauge needles. Parafilm was less effective than skin in reducing transmission of blood and viable mononuclear cells.

Acquired Immunodeficiency Syndrome↗
PubMed1977

Solvents, interfaces and protein structure.

Mean packing densities in protein interiors are comparable to those of most organic solids but the variations between small regions may be substantial. Packing defects may be related to allowed structural fluctuations. Molecular surface areas can be correlated with free energies of transfer between different solvents. The proportionality factor will depend, in general, on the nature of the solute and both the solvents. The changes in solvent-protein interfacial area on chain folding are large and the implied changes in free energy from this solvent-squeezing effect are correspondingly large. The strong tendency to minimize surface area is reflected in the globular shape of most protein molecules or domains in larger structures. The formation of isolated units of secondary structure from an extended chain represents about one half of the eventual total area change. The tendencies of amino acids to form beta-sheets correlate well with the rank-ordered list based on non-polar area change for each residue type. The calculated area changes for helix and sheet formation are not identical in rank order. The rank-ordered list for alpha-helix formation correlates satisfactorily with the probability list prepared from actual structures if glutamic acid and tyrosine are removed. What special characteristics unrelated to surface area these two amino acids might have is not clear. Tertiary structure formation from preformed secondary structural units can be rank ordered on area change and possible nucleation sites can be identified. A prediction scheme for helix-helix interactions is proposed. The hydrophobic force begins to be felt when two helices are about 0.6 nm (6 A) from their final contact positions. Interfacial surface tension is a logical parameter to relate free energy and solvent contact area, but this macroscopic parameter must be used with great caution. It is suggested that water in the deep grooves, characteristic of the active sites of many enzymes, may have a substantially higher fugacity than bulk water as indicated, at least qualitatively, by the Kelvin equation based on surface curvature. Such water would be more easily displaced than its plane surface counterpart and could contribute significantly to ligand-binding energy. This factor would be in addition to the usual solvent entropic effects associated with surface area reduction on association.

Amino Acids↗
PubMed2006

Cytidine 5'-monophosphate (CMP)-induced structural changes in a multifunctional sialyltransferase from Pasteurella multocida.

Sialyltransferases catalyze reactions that transfer a sialic acid from CMP-sialic acid to an acceptor (a structure terminated with galactose, N-acetylgalactosamine, or sialic acid). They are key enzymes that catalyze the synthesis of sialic acid-containing oligosaccharides, polysaccharides, and glycoconjugates that play pivotal roles in many critical physiological and pathological processes. The structures of a truncated multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1), in the absence and presence of CMP, have been determined by X-ray crystallography at 1.65 and 2.0 A resolutions, respectively. The Delta24PmST1 exists as a monomer in solution and in crystals. Different from the reported crystal structure of a bifunctional sialyltransferase CstII that has only one Rossmann domain, the overall structure of the Delta24PmST1 consists of two separate Rossmann nucleotide-binding domains. The Delta24PmST1 structure, thus, represents the first sialyltransferase structure that belongs to the glycosyltransferase-B (GT-B) structural group. Unlike all other known GT-B structures, however, there is no C-terminal extension that interacts with the N-terminal domain in the Delta24PmST1 structure. The CMP binding site is located in the deep cleft between the two Rossmann domains. Nevertheless, the CMP only forms interactions with residues in the C-terminal domain. The binding of CMP to the protein causes a large closure movement of the N-terminal Rossmann domain toward the C-terminal nucleotide-binding domain. Ser 143 of the N-terminal domain moves up to hydrogen-bond to Tyr 388 of the C-terminal domain. Both Ser 143 and Tyr 388 form hydrogen bonds to a water molecule, which in turn hydrogen-bonds to the terminal phosphate oxygen of CMP. These interactions may trigger the closure between the two domains. Additionally, a short helix near the active site seen in the apo structure becomes disordered upon binding to CMP. This helix may swing down upon binding to donor CMP-sialic acid to form the binding pocket for an acceptor.

Crystallization↗
PubMed1996

Synthesis and X-ray crystal structure of trans,cis-[Pt(OAc)2I2(en)]: a novel type of cisplatin analog that can be photolyzed by visible light to DNA-binding and cytotoxic species in vitro.

An original approach intended to facilitate the intratumoral activation of Pt(IV) diamines by illumination with visible light to form photolysis products that irreversibly bind to DNA and are cytotoxic to human cancer cells is reported. The novel Pt(IV) complex trans,cis-[Pt(OAc)2I2-(en)] was prepared by the acetylation of trans,cis-[Pt(OH)2I2(en)] with acetic anhydride in CH2-Cl2; trans,cis-[Pt(OH)2I2(en)] was synthesized by oxidation of [PtI2(en)] with 30% aqueous H2O2. trans,cis-[Pt(OAc)2I2(en)] crystallized from methanol as deep-red needles with a = 9.029(4) A, b = 11.443(2) A, c = 12.822(2) A, beta = 95.48(3) degrees, monoclinic space group Cc, and Z = 4. The conformation of the acetato groups around the O-Pt-O axis deviated significantly from the conformation of the acetato groups in the X-ray crystal structure reported for the cis-dichloro analog, which may explain the very different aqueous solubilities of the two compounds. trans,-cis-[Pt(OAc)2I2(en)] and trans,cis-[Pt(OH)2I2(en)] displayed broad ligand-to-metal charge-transfer bands centered at lambda = 389 and 384 nm, respectively (epsilon = 1372 and 1425 M-1 cm-1, respectively), with tailing out to ca. 550 nm. When trans,cis-[Pt(OAc)2I2(en)] was incubated with calf thymus DNA in the absence of light, no covalent binding of Pt to DNA was measurable after 6 h; however, irradiation with light of wavelengths > 375 nm resulted in 63 +/- 13% of the platinum being covalently bound to DNA after 6 h, suggesting that a photoreduction to Pt(II) species took place. Although trans,cis-[Pt(OH)2I2(en)] was also labile to visible light, only 10 +/- 2% DNA platination was observed after 6 h of illumination; however, covalent binding of Pt to DNA took place quantitatively when a reducing agent such as glutathione was added to the photolyzed incubations. These results provide evidence that the photolysis of the trans-dihydroxo analog resulted predominately in the substitution of the iodide ligands for water rather than a reduction of Pt(IV) to Pt(II). When protected from light, trans,cis-[Pt(OAc)2I2-(en)] and trans,cis-[Pt(OH)2I2(en)], both at a concentration of 10 microM, had half-lives of 6.6 +/- 0.5 and 46.8 +/- 8.8 h, respectively, at 37 degrees C in Eagle's minimum essential medium (EMEM) containing 5% fetal calf serum. When irradiated with light lambda(irr) > 375 nm, the half-lives were decreased by 24- and 53-fold for the diacetato- and dihydroxoplatinum(IV) complexes, respectively. Compared to the "dark" control, the in vitro treatment of TCCSUP human bladder cancer cells with trans,cis-[Pt(OAc)2I2(en)] resulted in 35% greater growth inhibitory activity when during the first 1.5 h of drug exposure the cells were irradiated with light lambda irr > 375 nm. The photolysis of trans,cis-[Pt(OH)2I2(en)] with visible light resulted in a 22% enhancement of antiproliferative activity.

Antineoplastic Agents↗
PubMed2004

Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies.

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively. MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively. Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03], which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonation of the thiolate-imidazolium ion. In addition, substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered to rapidly form a stable acetylated species poised to react with an arylamine substrate.

Acetamides↗