Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase

Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.     

Role of the disulfide bond in Shiga toxin A-chain for toxin entry into cells

Shiga toxin consists of an enzymatically active A-chain and a pentameric binding subunit. The A-chain has a trypsin-sensitive region, and upon cleavage two disulfide bonded fragments, A1 and A2, are generated. To study the role of the disulfide bond, it was eliminated by mutating cysteine 242 to serine. In T47D cells this mutated toxin was more toxic than wild type toxin after a short incubation, whereas after longer incubation times wild type toxin was most toxic. Cells cleaved not only wild type but also mutated A-chain into A1 and A2 fragments. The mutated A-chain was more sensitive than wild type toxin to Pronase, and it was degraded at a higher rate in T47D cells. Subcellular fractionation demonstrated transport of both wild type and mutated toxin to the Golgi apparatus. Brefeldin A, which disrupts the Golgi apparatus, protected not only against Shiga toxin but also against the mutated toxin, indicating involvement of the Golgi apparatus. After prebinding of Shiga(C242S) toxin to wells coated with the Shiga toxin receptor, Gb3, trypsin treatment induced dissociation of A1 from the toxin-receptor complex demonstrating that in addition to stabilizing the A-chain, the disulfide bond prevents dissociation of the A1 fragment from the toxin-receptor complex.     

Rapid detection of Shiga-like toxin gene in feces with rapid-cycle polymerase chain reaction

A rapid detection for Shiga-like toxin in feces was developed with the nucleic acid extraction method by silicondioxide-guanidine thiothianate and rapid-cycle polymerase chain reaction by RapidCycler (model 1002; Idaho Technology, RC-PCR here after). Twenty-two fecal samples that were collected from patients with diarrhoea caused by E. coli O157:H7 and frozen for 6 months were examined directly by RC-PCR, conventional PCR assay using by ThermalCycler 9600-R (Roche, TC-PCR here after) and by the culture method using tellurite-cefixime sorbitol MacConkey (direct method). These examinations were done also after being injected into TCV-TSB and incutated at 35 degrees C overnight (indirect method). The sensitivity of RC-PCR and TC-PCR using a diluted suspension of broth enriched at 35 degrees C overnight were 4.1 pg and 410 fg, respectively. Positive results in the direct method were obtained in 7 for RC-PCR, 10 for TC-PCR and 5 for culture. Positive results on indirect assay were obtained in 9 for RC-PCR, 9 for TC-PCR and 7 for culture. It was demonstrated that the RC-PCR assay was able to detect Shiga-like toxin gene in feces in less than 90 minutes after being received at the laboratory.     

The calcium-binding sites of heparinase I from Flavobacterium heparinum are essential for enzymatic activity

In the accompanying paper (Shriver, Z., Liu, D., Hu, Y., and Sasisekharan, R. (1999) J. Biol. Chem. 274, 4082-4088), we have shown that calcium binds specifically to heparinase I and have identified two major calcium-binding sites (CB-1 and CB-2) that partly conform to the EF-hand calcium-binding motif. In this study, through systematic site-directed mutagenesis, we have confirmed the accompanying biochemical studies and have shown that both CB-1 and CB-2 are involved in calcium binding and enzymatic activity. More specifically, we identified critical residues (viz. Asp210, Asp212, Gly213, and Thr216 in CB-1 and Asn375, Tyr379, and Glu381 in CB-2) that are important for calcium binding and heparinase I enzymatic activity. Mutations in CB-1 resulted in a lower kcat, but did not change the product profile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the kcat for heparinase I, but also resulted in incomplete degradation, leading to longer saccharides. Fluorescence competition experiments along with heparin affinity chromatography suggested that mutations in CB-1 alter heparinase I activity primarily through decreasing the enzyme's affinity for its calcium cofactor without altering heparin binding to heparinase I. Compared with CB-1 mutations, mutations in CB-2 affected calcium binding to a lesser extent, but they had a more pronounced effect on heparinase I activity, suggesting a different role for CB-2 in the enzymatic action of heparinase I. These results, taken together with our accompanying study, led us to propose a model for calcium binding to heparinase I that includes both CB-1 and CB-2 providing critical interactions, albeit via a different mechanism. Through binding to CB-1 and/or CB-2, we propose that calcium may play a role in the catalytic mechanism and/or in the exolytic processive mechanism of heparin-like glycosaminoglycan depolymerization by heparinase I.     

Diarrhea in young children associated with Escherichia coli non-O157 organisms that produce Shiga-like toxin

The aim of this study was to evaluate the way in which short-term protection declines and is eventually lost in preconditioning and to determine the efficacy of a second preconditioning at various reperfusion intervals. Male rabbits were divided into six groups. Forty-five minutes (sustained) ischemia followed by 120 minutes reperfusion was applied 60, 65, 70, 75, and 80 minutes after a 5 minute preconditioning (groups A, B, C, D, and E) and in a control group (F) after no preconditioning. The infarct to risk ratio (I/R) was 38.3 +/- 3.5% in group A, 46.0 +/- 7.8% in B, 61.6 +/- 9.7% in C, 68.1 +/- 4.2% in D, 64.5 +/- 7.8% in E, and 61.0 +/- 7.7% in F. Group A had a smaller I/R compared with groups C, D, E, and F (p < 0.05). In another series, groups G, H, and I were exposed to two 5-minute preconditioning stimuli, separated, respectively, by 45, 60, and 75 minutes of reperfusion; 10 minutes after the last preconditioning, the animals were exposed to 45-minutes ischemia and 120 minutes reperfusion. Groups A and D (with the smaller and higher I/R ratio) were also incorporated into this protocol in order to compare the effect of the additional preconditioning with the single one. The I/R ratio was 25.4 +/- 8.5% in group G, 22.8 +/- 7.0% in group H, and 14.7 +/- 4.0% in group I (p = NS). Group D showed a higher I/R compared with groups G, A, and H (p < 0.01), and group I had a smaller I/R compared with groups A (p < 0.01) and D (p < 0.001). Cardioprotection after a first preconditioning declines gradually and is eventually lost. An additional preconditioning is always effective, and the longer the interval from the first preconditioning, the more potent is the effect.     

Escherichia coli outer membrane phospholipase A: role of two serines in enzymatic activity

In the outer membrane phospholipase A (OMPLA) of Escherichia coli, Ser144 has previously been identified by chemical modification as the active site serine residue. In a specific OMPLA-negative mutant strain, the pldA gene coding for OMPLA was shown to differ from the wild-type gene by a single point mutation, resulting in the substitution of Ser152 by phenylalanine. The role in catalysis of these two serine residues in OMPLA was investigated by site-directed mutagenesis. Ser144 and Ser152 were replaced one at the time by either alanine, valine, phenylalanine, threonine, or cysteine. Ser152 was furthermore replaced by asparagine. Replacement of Ser144 by cysteine resulted in 1% residual activity, whereas the other substitutions at this position yielded virtually inactive enzymes. Substitution of Ser 152 by threonine or asparagine resulted in 40% and 2% residual activity respectively, whereas all other substitutions at this position resulted in the loss of enzymatic activity. We propose that Ser144 is the nucleophile in catalysis, and that Ser152 is involved in hydrogen bonding either to the catalytic triad or in the oxyanion hole.     

Low-level release of Shiga-like toxin (verocytotoxin) and endotoxin from enterohemorrhagic Escherichia coli treated with imipenem

Shiga-like toxin (SLT) and endotoxin may participate in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC) infection. Levels of release of SLT and endotoxin from EHEC treated in vitro with antibiotics were estimated. There were differential levels of release of SLT and endotoxin from EHEC treated with different antibiotics. Treatment of EHEC strains, namely, E. coli O157, O111, and O26, with imipenem induced much lower levels of release of SLT and endotoxin than treatment with ceftazidime.     

The role of the basal ganglia in control of motor activity

Morphological, histochemical and functional analysis of the role of basal ganglia in the motor control is presented. The inhibiting effect of basal ganglia on the motor activity, their role in programming the slow and stereotyped movements, and the presentation of emotional and memory centres to the motor mechanisms of behaviour is emphasised.     

An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA

1. We describe the first application of microdialysis to monitor the pharmacokinetics of a drug in the blood of man. 2. The aims of the study were to ascertain patient acceptability and tolerability of a new microdialysis probe and to assess its accuracy in determining the pharmacokinetics of levodopa and its principal plasma metabolite 3-O-methyldopa (3-OMD). 3. Eight patients with parkinsonism on chronic levodopa therapy were investigated. 4. After an overnight fast, a flexible microdialysis probe, perfused with isotonic saline, was inserted into a forearm vein and a blood sampling cannula was inserted in a forearm vein of the other arm. After ingestion of a levodopa preparation (Madopar Dispersible), dialysate was collected over 5 or 10 min periods and blood samples were taken every 15 or 30 min for 2-6 h. 5. Dialysate drug profiles were similar to those of plasma, and levodopa and 3-OMD concentrations exhibited significant (P < 0.001) correlation with those observed in the corresponding plasma samples. 6. The mean (+/- s.d.) blood dialysate concentrations for levodopa and 3-OMD were 36.1 +/- 9.2% and 43.4 +/- 8.4% respectively of the plasma content. 7. The tolerability of the probe was excellent, and all eight patients found it preferable to conventional blood sampling. 8. Microdialysis of blood is less invasive than frequent intermittent direct blood sampling, and can readily be used to continuously monitor levodopa pharmacokinetics. In a clinical setting, a combination of drug monitoring by this technique together with clinical evaluation of motor function can be used to optimize levodopa treatment in patients with Parkinson's disease.     

The relationship between insulin bioactivity and structure in the NH2-terminal A-chain helix

Old females are compared to young females for the purpose of studying the difference in comfort caused by the environmental variables of temperature and humidity as well as the form of clothing. Eight experiments were performed in three settings: (a) 30 degrees C R.H.80%; 30 degrees C R.H.45%; and 20 degrees C R.H.45%. The ages of the subjects range from 62 to 68 (Mean = 65.17, S.D. = 1.68) among old females and from 20 to 23 (Mean = 20.83, S.D. = 0.76) among young females. The following results were obtained: (1) The young females were sensitive to hot temperatures, while the old females were not. On the other hand, the old females were more sensitive to cold temperatures, under 20 degrees C R.H.45%, than the young females. In temperatures under 30 degrees C R.H.80%, the heat radiation from the young females was higher than that of the old females. Under 20 degrees C R.H.45%, the heat radiation from the old females was higher than from the young females. The old females are thought to decline in physiogenic function due to enduring both hot and cold temperatures. (2) The correlation between the temperature in clothes and comfort among the old females is not different from the same correlation among the young females. This conclusion agrees with previously published studies of the young females. (3) Skin temperature and bloodstream are measured, according to clothing form. As a result, a long skirt is the highest in thermal insulation, long pants the next highest, and a short skirt is the lowest. (4) The effect of thermal insulation provided by a lap robe was tested in both groups. The lap robe was found to be more effective for the older group than the younger in temperatures under 20 degrees C R.H.45%. Hence, the role of clothes in offsetting for the decline in the thermoregulation function that compensates for environmental change is more important for old females than for young.     

Antitumor effect of diphtheria toxin A-chain gene-containing cationic liposomes conjugated with monoclonal antibody directed to tumor-associated antigen of bovine leukemia cells

Monoclonal antibody c143 against tumor-associated antigen (TAA) expressed on bovine leukemia cells was conjugated to cationic liposomes carrying a plasmid pLTR-DT which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) in the multicloning site of pUC-18. The specificity and antitumor effects of the conjugates were examined in vitro and in vivo using TAA-positive bovine B-cell lymphoma line as the target tumor. In vitro studies with the TAA-positive cell line indicated that luciferase gene-containing cationic liposomes associated with the c143 anti-TAA monoclonal antibody caused about 2-fold increase in luciferase activity compared with cationic liposomes having no antibody, and also that the c143-conjugated cationic liposomes containing pLTR-DT exerted selective growth-inhibitory effects on the TAA-positive B-cell line. Three injections of pLTR-DT-containing cationic liposomes coupled with c143 into tumor-bearing nude mice resulted in significant inhibition of the tumor growth. The antitumor potency of the c143-conjugated cationic liposomes containing pLTR-DT was far greater than that of normal mouse IgG-coupled cationic liposomes containing pLTR-DT as assessed in terms of tumor size. These results suggest that cationic liposomes bearing c143 are an efficient transfection reagent for BLV-infected B-cells lymphoma cells, and that the delivery of the pLTR-DT gene into BLV-infected B-cells by the use of such liposomes may become a useful technique for gene therapy of bovine leukosis.     

Resistance of purified cholera toxin to enzymatic treatment with pancreatic elastase and papain

Treatment of Cholera toxin with pancreatic elastase and papain in vitro showed a high resistance of the toxin molecule to these enzymes, under non-denaturing conditions or in the presence of 2 M urea. These experiments support the hypothesis of a particularly stable molecular structure of the toxin, as an explanation of its activity in the intestinal lumen where the pancreatic proteases are active.     

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non-E. coli bacteria. Its detection limit was 10(2)-10(3) cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10(0) cells 100 ml-1 of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

Histology and enzymatic activity in the postnatal development of limb muscles in rodents

The present work examines how increases in spontaneous motor capabilities during postnatal development are reflected in enzymatic activity and the histology of hindlimb muscles of the dormouse (Eliomys melanurus), the jird (Meriones tristrami), the vole (Microtus socialis), and the spiny mouse (Acomys cahirinus). The precocial neonate of the spiny mouse had the most advanced developmental state of young myofibers with striations as early as 1 week after delivery. At the same age, the altricial neonate vole had less developed muscles compared to the spiny mouse, but was more mature compared to other altricial species. The dormouse was the least developed, with numerous myoblasts and few myotubes at 1 week after delivery. These differences in myogenic development were conspicuous throughout postnatal development. Similar differences between the species were also evident at the biochemical level, as measured in the kinetics of activity of the enzyme creatine-phosphokinase immediately after delivery. On postnatal day 7, the creatine-phosphokinase level in the spiny mouse was fourfold higher than in the dormouse or vole. The enzymatic activity of acid phosphatase decreased during the first week postdelivery in the spiny mouse while peaking in the first, second, and third week in the jird, vole, and dormouse, respectively. These results support the notion that precocial species undergo certain developmental stages in utero, whereas, the same stages commence in altricials only postnatally. For the tested altricial species, the results illustrate that limb muscles in the vole, which displays more basic gaits, mature before limb muscles of the jird and dormouse, which display more specialized gaits.     

DNA synthesis enzymatic activity in the bone marrow of rats of different ages

The activity of DNA synthesis enzymes was studied in bone marrow of rats aging from 1 month to 2.5 years. The activity of enzymes of thymidine and thymidylic acid phosphorylation in the rat bone marrow is determined to be practically constant during the whole period of studies. Activity of gCMP-desaminase and DNA-polymerase has a tendency to a decrease during the life period of rats from one to six-eight months, being constant in the following periods.     

Retinal detachment following congenital cataract surgery. I. Preoperative findings in 114 eyes

One hundred fourteen eyes of patients with retinal detachment occurring after congenital cataract surgery were studied. Retinal detachment was typified by high incidences of men, myopia, preference for the second and fourth decades of life, and a fairly long interval after cataract surgery. Frequently found were the following: (1) undetected retinal breaks, (2) high incidences of small oval or round holes in the upper nasal quadrant near the ora serrata, (3) retinal detachment in more than one quadrant, and (4) extensive vitreous and preretinal traction. Preoperative examination was often hampered by a small, bound-down pupil, nystagmus, extreme photophobia, and an inability to move the eye in desired directions. The major factor in the pathogenesis of retinal detachment after congenital cataract surgery appears to be chronic vitreoretinal traction in the anterior vitreous caused by cataract removal.