Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.     

Dealing with errors in interactive sequencing by hybridization.

MOTIVATION: A realistic approach to sequencing by hybridization must deal with realistic sequencing errors. The results of such a method can surely be applied to similar sequencing tasks. RESULTS: We provide the first algorithms for interactive sequencing by hybridization which are robust in the presence of hybridization errors. Under a strong error model allowing both positive and negative hybridization errors without repeated queries, we demonstrate accurate and efficient reconstruction with error rates up to 7%. Under the weaker traditional error model of Shamir and Tsur (Proceedings of the Fifth International Conference on Computational Molecular Biology (RECOMB-01), pp 269-277, 2000), we obtain accurate reconstructions with up to 20% false negative hybridization errors. Finally, we establish theoretical bounds on the performance of the sequential probing algorithm of Skiena and Sundaram (J. Comput. Biol., 2, 333-353, 1995) under the strong error model. AVAILABILTY: Freely available upon request. CONTACT: skiena@cs.sunysb.edu.     

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.     

Rapid determination of antimicrobial susceptibility patterns ofListeria monocytogenes isolates by the autobac AIS and MIC procedures

A total of 34 isolates ofListeria monocytogenes were tested against ampicillin, cephalothin, chloramphenicol, erythromycin, tetracycline, and penicillin-G using the Autobac 3-h AIS and the Autobac 5-h MIC procedures. The results were compared to susceptibility category interpretations and MICs determined using the Sceptor system. With the Sceptor System, all isolates were interpreted to be moderately susceptible to ampicillin and penicillin-G, and susceptible to the four other antibiotics. With the Autobac AIS, all isolates were interpreted to be susceptible to all the antibiotics except penicillin-G. All but one of the 34 isolates were interpreted to be resistant to penicillin-G with the Autobac AIS test. The remaining isolate was interpreted to be indeterminant. The Autobac AIS test was unsatisfactory for determining the susceptibility ofL. monocytogenes isolates to penicillin-G. The Autobac MIC results correlated well with the MIC results of the Sceptor system provided that the Autobac was programmed as though it were testing enterococci. The Autobac MIC reported penicillin-G MICs in units per milliliter and required the use of a conversion factor to obtain micrograms per milliliter, and did not allow for the testing of erythromycin. The Autobac MIC susceptibility category interpretations must not be used, as they were derived from an outdated susceptibility standard. The Autobac MIC test may be used if the limitations given above are observed.     

Dynamics of Imbibition in Phaseolus vulgaris L. in Relation to Initial Seed Moisture Content

The seed moisture level marking the onset of imbibitional injury (breakpoint) was determined for two cultivars of Phaseolus vulgaris L. cvs `Tendercrop' (TC) and `Kinghorn Wax' (KW). At 20°C the breakpoints were 0.15 gram H₂O/gram dry weight (gram per gram) for TC and 0.11 gram per gram for KW. When seeds were imbibed at 5°C, the breakpoints were 0.19 gram per gram (TC) and 0.16 gram per gram (KW). Below the breakpoint germination changed 4.6%/0.01 gram per gram for all treatments. Imbibition rates were maximal at 0.07 gram per gram and 0.33 gram per gram after 20 minutes imbibition. Rates of electrolyte leakage were correlated with the imbibition rate maximum at 0.07 gram per gram but were unaffected by the maximum at 0.33 gram per gram. The transition from tightly bound to semibound water occurred at 0.09 gram per gram and 0.11 gram per gram for KW and TC, respectively. T1 values increased exponentially as seed moisture decreased from 0.47 gram per gram to 0.05 gram per gram. ¹³C-NMR sugar signals increased at moisture levels above 0.14 gram per gram and plateaued at approximately 0.33 gram per gram seed moisture. These results suggest that the breakpoint moisture level for imbibitional damage is a function of temperature while the injury process is similar at both 5 and 20°C. Imbibition and leakage rate maxima reflect transitions in the states of seed water. NMR data support the application of the Water Replacement Hypothesis to seeds. Thus, imbibitional injury may be related to specific, temperature dependent moisture levels that are determined by water binding characteristics in the seed tissue.     

Construction of miniplasmids from the 7.2-kb and 5.1-kb penicillinase-producing plasmids of Neisseria gonorrhoeae reveals two replication regions

Two replication regions have been identified on a 7.2-kb penicillinase-producing plasmid (pJD4) of Neisseria gonorrhoeae. Through construction of mini-plasmids, one replication region of pJD4 was located on a 1.5-kb fragment, designated region "a," that included the unique HindIII site of this plasmid. This region is absent from the 5.1-kb naturally occurring gonococcal penicillinase-producing plasmid (pJD5) which is considered to be a deletion-derivative of the 7.2-kb plasmid. A 1.5-kb fragment (region "b"), part of a 2.5-kb fragment essential for the replication of the 5.1-kb plasmid (pJD5), was found to be responsible for incompatibility. Incompatibility studies showed that in vitro-derived deletion-derivatives from pJD4 and pJD5 containing either region "a" or region "b" were compatible. The DNA sequence of part of region "a" showed that this region was A-T rich. It contained seven sets of A-T rich multiple direct repeats and two putative dnaA boxes, suggesting that the mechanism of replication of region "a" was similar to that of OriC in Escherichia coli.     

Platelet immune complex interaction in pathogenesis of Kawasaki disease and childhood polyarteritis.

The role of platelets in the pathogenesis of vasculitis and the formation of coronary artery aneurysms was studied in 19 children with Kawasaki disease and five with polyarteritis. All patients with Kawasaki disease developed thrombocytosis in the third week of illness. The peak platelet count was significantly correlated (p less than 0.005) with the subsequent development of coronary artery aneurysms. The rise in platelet count was associated with the appearance in the circulation of a factor that induced aggregation and serotonin release in normal platelets. This factor was shown to be of high molecular weight, and its activity was lost at low pH--features suggestive of an immune complex. Immune complexes, detected by precipitation with polyethylene glycol, also appeared in the circulation as the platelet count increased. These complexes induced platelet aggregation, and there was a significant correlation (p less than 0.001) between the concentrations of IgG and IgA in the polyethylene glycol precipitated material and the platelet aggregating activity. Similar platelet aggregating activity was also detected in patients with polyarteritis but followed a different time course, persisting in the circulation for several months in association with continued disease activity. These findings imply that different mechanisms have a role in distinct phases of Kawasaki disease. The initial feverish phase (probably infective) is probably followed by an immune complex vasculitis that occurs when antibodies to the initiating agent appear in the circulation. The immune complexes aggregate platelets and induce release of serotonin. Platelet derived vasoactive mediators may increase vascular permeability and facilitate further deposition of complexes in the tissues.     

Possible metabolic basis for the developmental anomaly observed in in vitro culture,called ‘vitreous plants’

Previous studies have shown that the development anomaly encountered in meristem culture, known as vitreous plants, is due to deficient lignin synthesis. This anomaly can be cured by addition of phloridzin to the culture medium. This study examines the activities of some enzymes involved in the synthesis of lignins and of flavonoids in normal and in vitreous plants of two apple cultivars. The results showed that all enzymes were consistently less active in the vitreous plants. This agrees with previous studies made on the hydroxycinnamate: CoA ligase activity in Prunus avium (L.) meristem-derived plants. The study on the substrate specificity of the enzyme demonstrates that while its activity is lower in the vitreous plant, its conformation is identical with that of the normal plant; the substrate that is specific to enzyme extracts of both sources is para-coumaric acid.     

3H]spiroperidol binding on lymphocytes: changes in two different groups of schizophrenic patients and effect of neuroleptic treatment

[3H]spiroperidol binding to lymphocytes was measured in untreated paranoid or disorganized and treated paranoid schizophrenic patients. An increase in the Bmax was detected in untreated paranoid patients but a decrease was found in the disorganized patients. No difference was detected in the KD value. Neuroleptic treatment produced a decrease in the Bmax without affecting the KD value. Such results did not comply with the down regulation but might be explained by a change in membrane viscosity as [3H]spiroperidol binding sites on lymphocytes were coupled to phospholipid methylation.