Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY

The bacterial chemotaxis protein CheY is activated in vivo by the covalentphosphorylation of a single aspartate residue at position 57. However, thisphosphate linkage is unstable (t1/2 approximately 20 s at roomtemperature), thereby precluding many biochemical analyses. Here we presenta synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) withchemical stability of the phosphate linkage enhanced by several orders ofmagnitude relative to the native protein. Starting with CheY D57C, asite-specific mutant of CheY with a unique cysteine residue in place of theaspartate at position 57, two sequential disulfide exchange reactions wereperformed to form the final product 'CheY D57C-SPO3' with a thiophosphatemoiety covalently bonded to the protein in a disulfide linkage. Massspectral analysis showed that the desired analog was present at 70-80% ofthe total protein. The disulfide linkage had a t1/2 of 8 days at 4 degreesC. Biochemical characterization of CheY D57C-SPO3 included assessment ofconformational properties using tryptophan fluorescence, evaluation ofmetal binding properties and measurement of binding interactions with thechemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group ata nearly identical location as native CheY-phosphate, the analog was unableto emulate CheY-phosphate function, thereby supporting the idea that thereare very precise geometric requirements for successful CheY activation.     

Full-arch implant framework casting accuracy: preliminary in vitro observation for in vivo testing

PURPOSE: Conventional techniques for implant metal framework fabrication produce error of a magnitude that is inconsistent with the passive-fit requirement for osseointegrated implants. To understand the correlation between prosthesis fit and the implant-tissue response, evaluation of the interface tissue reactions to customary levels of fit is required. The purpose of this study is to determine the accuracy of torch casting full arch frameworks using a high palladium alloy and a ringless phosphate-bonded investment technique. MATERIALS AND METHODS: Three different variables were considered relative to casting accuracy effect. The first variable, completeness of mold-fill, compared cast specimens where the entire sprue system was filled as part of the casting and cast specimens without the sprue system filled. The second variable, phosphate-bonded investment special liquid concentrations, compared groups of castings produced from 0%, 12%, 25%, and 50% special liquid. The third variable, investment mold shape, compared casting produced from a conventional ringless mold shape with a modified ringless mold shape where the investment in the same horizontal plane as the pattern was equal in thickness at the internal and external surfaces. Horizontal and vertical distances on the wax pattern and resulting framework were measured using a machinists microscope to determine casting error. Combined vertical and horizontal error was used for comparison between groups (one-way analysis of variance). RESULTS: No significant differences existed among the three groups compared (P > 0.05). The mean error comparison between the complete and incomplete mold-fill groups showed no statistical difference, while the incomplete fill group was found to be more porous. The mean error of all groups (0.130 mm) exceeded the recommended level of fit needed to satisfy the passive fit requirement by more than 10-fold. CONCLUSIONS: These results verify clinical observation and suggest that the use of conventional lost wax casting technique to cast one-piece full arch implant frameworks is both imprecise and inaccurate as judged against the passive fit requirement. The consequences of screw-fastening misfitting prostheses to osseointegrated implants is currently under investigation.     

Protein analysis of human von Ebner saliva and a method for its collection from the foliate papillae

The lingual serous glands of von Ebner are located close to the foliate and circumvallate papillae. Saliva secreted by these glands provides the immediate environment of the taste buds, and it has been hypothesized that it modulates taste perception. The purpose of this study was to develop a technique for collection of unstimulated and stimulated saliva from human von Ebner glands. Saliva was collected under resting conditions and after application of various gustatory stimuli (sweet, sour, salt, and bitter) by insertion of periostrips into the folds of the foliate papillae of healthy human volunteers. Stimulated saliva was also collected in glass microcapillaries or micropipettes. The flow-rates of unstimulated von Ebner saliva were 2.3 +/- 0.6 (S.E.) microL/min and 4.5 +/- 1.2 (S.E.) microL/min with 1% citric acid stimulation. The protein content was 2.5 +/- 0.5 (S.E.) mg/mL. The SDS gel electrophoretic profile of von Ebner saliva revealed two protein bands of Mr 18,000 that were identified on Western blots as von Ebner gland (VEG) proteins. Although lingual lipase activity was detected at very low levels by enzyme assay, this protein was not detected on Western blots. This collection technique should prove useful for analysis of specific functions associated with secretion from von Ebner glands.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

Effects of phentermine on responding maintained under multiple fixed-ratio schedules of food and cocaine presentation in the rhesus monkey

Drugs that decrease drug-maintained responding at doses that do not decrease other behaviors in animals may be suitable candidates for development as medications to treat drug abuse in humans. The present study examined whether this effect could be obtained with phentermine, a drug that has been reported to decrease cocaine intake in humans. Rhesus monkeys were trained under multiple fixed-ratio 30-response schedules of food and i.v. cocaine delivery. Phentermine was always given as a slow, i.v. infusion. Acute treatment with phentermine (0.3-10 mg/kg) decreased cocaine-maintained responding at doses that did not decrease, or decreased less, food-maintained responding for each of three unit doses of cocaine (10-100 microg/kg/injection). Subacute treatment with phentermine (3 or 5.6 mg/kg, daily) also decreased cocaine-maintained responding more than food-maintained responding. After subacute treatment was terminated, rates of cocaine-maintained responding generally recovered to levels comparable to those seen during untreated control sessions. Phentermine (0.3-3 mg/kg) did not generally increase responding associated with a very low (1 microg/kg/injection) unit dose of cocaine, suggesting that the decrease in cocaine-maintained responding at higher unit doses was not the result of a leftward shift in the cocaine unit dose-effect function. Phentermine (0.1-3 mg/kg) decreased responding maintained by 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909) (30 microg/kg/injection) at doses similar to those that decreased food-maintained responding. These results show that phentermine is effective in decreasing cocaine self-administration and suggest that it may be an effective medication for cocaine abuse.     

Acidic urine pH is associated with elevated levels of free urinary benzidine and N-acetylbenzidine and urothelial cell DNA adducts in exposed workers

We evaluated the influence of urine pH on the proportion of urinary benzidine (BZ) and N-acetylbenzidine present in the free, unconjugated state and on exfoliated urothelial cell DNA adduct levels in 32 workers exposed to BZ in India. Postworkshift urine pH was inversely correlated with the proportions of BZ (r = -0.78; P < 0.0001) and N-acetylbenzidine (r = -0.67; P < 0.0001) present as free compounds. Furthermore, the average of each subject's pre- and postworkshift urine pH was negatively associated with the predominant urothelial DNA adduct (P = 0.0037, adjusted for urinary BZ and metabolites), which has been shown to cochromatograph with a N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine adduct standard. Controlling for internal dose, individuals with urine pH < 6 had 10-fold higher DNA adduct levels compared to subjects with urine pH > or = 7. As reported previously, polymorphisms in NAT1, NAT2, and GSTM1 had no impact on DNA adduct levels. This is the first study to demonstrate that urine pH has a strong influence on the presence of free urinary aromatic amine compounds and on urothelial cell DNA adduct levels in exposed humans. Because there is evidence that acidic urine has a similar influence on aromatic amines derived from cigarette smoke, urine pH, which is influenced by diet, may be an important susceptibility factor for bladder cancer caused by tobacco in the general population.     

A study on expression of the heat shock suppressed gene in distal organs of rats after scalding

In order to address the question whether stress in intact higher animals may induce cellular heat shock response in distal organs, the inhibition of normal gene expression was studied on the basis of our previous findings about the induction of heat shock proteins in liver and brain of rats after scalding. Male SD rats were scalded on the back, 10-240 min thereafter decapitated, and the heat shock suppressed gene-1 was quantitated by dot blotting. The results showed that gene-1 decreased rapidly after scalding in both the organs, and did not recover to control levels even 240 min after scalding. The decrease of gene-1 went parallell with the severity of scalding. Thus it may be concluded that stress may induce heat shock response of distant organs in intact animals. Possible pathological significance of these findings was discussed.