Biochemical identification of transmembrane segments of the Ca(2+)-ATPase of sarcoplasmic reticulum

The transmembrane segments of sarcoplasmic reticulum Ca(2+)-ATPase were determined by trypsinization of cytoplasmic side-out intact sarcoplasmic reticulum vesicles. The membrane portion of tryptic digest comprising the transmembrane fragments, joined by the intravesicular segments, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after labeling with fluorescein 5-maleimide in the presence of sodium dodecyl sulfate. In this way, seven fluorescent bands of tryptic fragments below 11 kDa were observed which were derived from 4 pairs of membrane spanning segments and one hydrophobic sequence at the C-terminal end. Two peptides of 10.8 and 10.6 kDa had the identical N-terminal sequence beginning at Glu826, representing the transmembrane segments M7 and M8 and their connecting loop. A band at 8.1 kDa contained one peptide beginning at Tyr36 (M1/loop/M2). A 7.7-kDa peptide starting at Leu253 (M3/loop/M4) and a 7.3-kDa peptide beginning at Ala752 (M5/loop/M6) were also observed. A band at 6.7 kDa contained two peptides, one beginning at Ser48 (M1/loop/M2) and another beginning at Tyr763 (M5/loop/M6). In addition, a 4-kDa peptide beginning at Met925 was observed. The size of this peptide did not allow for a complete pair of transmembrane segments, but this peptide could have been derived from trypsinolysis between the last pair of membrane spanning segments. These data therefore provide biochemical evidence for at least 8 transmembrane segments and perhaps two more at the C-terminal end of the enzyme.     

On regional nonlinear H-filtering

The structure of the nonlinear H^∞-filter in the neighborhood of the estimated trajectory is investigated and a bound on the size of the neighborhood that allows this structure is determined, both for finite and infinite horizons. Riccati inequalities that depend on the estimated trajectory are derived for finding the filter gain matrix and an algorithm for calculating the bound on the size of the above neighborhood is presented. Explicit formulas are obtained in the infinite horizon case for the minimum achievable disturbance attenuation level, the size of the neighborhood, and the corresponding filter gain.     

Alternative measures of pesticide use

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependent methylation of uridine-54 in the T psi C-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried and the question arises as to how RUMT gains access to the methylation site. A 17-mer RNA hairpin consisting of nucleotides 49-65 of the T psi-loop is a substrate for RUMT. Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD) methods were used to determine the solution structure of the 17-mer T-arm fragment. The loop of the hairpin exhibits enhanced flexibility which renders the conventional NMR average structure less useful compared to the more commonly found situation where a molecule exists in predominantly one major conformation. However, when resorting to softer refinement methods such as MD with time-averaged restraints, the conflicting restraints in the loop can be satisfied much better. The dynamic structure of the T-arm is represented as an ensemble of 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the T psi-loop in solution in conjunction with extensive binding studies of RUMT with the T psi C-loop and tRNA suggest that the specificity of the RUMT/ tRNA recognition is associated with tRNA tertiary structure elements. For the methylation, RUMT would simply have to break the tertiary interactions between the D- and T-loops, leading to a melting of the T-arm structure and making U54 available for methylation.     

Transrectal electrostimulation therapy for neuropathic bowel dysfunction in children with myelomeningocele

PURPOSE: We attempted to evaluate the efficacy of transrectal bowel stimulation for neurogenic bowel dysfunction in children with myelodysplasia. MATERIALS AND METHODS: Daily sessions of transrectal electrostimulation were performed on an outpatient basis for 2 to 3 weeks on children with myelodysplasia and stool incontinence. If benefits were noted, 5 to 10 additional daily sessions were performed. Complete success was defined as improvement in all parameters of interest, including decrease in the frequency of daily bowel movements, increased sensation, increased ability to hold stool and a significant subjective change in bowel habits. Moderate success implied improvement in 1 to 3 parameters and treatment failure was defined as lack of improvement in any parameter. RESULTS: A total of 55 children 2 to 14 years old (mean age 6.7) completed a mean of 18 daily sessions per patient of bowel electrostimulation. Followup ranged from 1 to 6 years. Diapers are no longer required due to defecation problems in 14 children older than 3 years. Complete success was achieved in 20 cases (36.3%) and moderate success in an additional 30 (54.5%, overall success rate 90.8%). Specifically, 89% of the patients reported elimination of stooling accidents, 82% reported increased sensation and 71% were able to hold the bowel movement. Overall 68% of the patients noticed significantly improved bowel function. Complete/moderate success of transrectal electro-stimulation was statistically significant for all 4 parameters (p < 0.05), and complete success was significant for increased sensation, ability to hold and episodes of accidents. Therapy failed in 5 children (9%). There were no untoward effects. CONCLUSIONS: Transrectal electrostimulation is a well tolerated and minimally invasive modality that provides sustainable improvement in stool continence in children with myelomeningocele and neuropathic bowel dysfunction.     

Detection of melanoma by monitoring the intracellular fluorescein fluorescence polarization changes in lymphocytes

The effectiveness of detecting melanoma by measuring the intracellular fluorescein fluorescent polarization (IFFP) of patients' SCM (structuredness of the cytoplasmic matrix)-responding lymphocytes was examined. SCM-responding lymphocytes from 46 melanoma patients and 32 healthy volunteers were labeled with fluorescein diacetate and challenged with different stimuli, and the resulting polarization was determined. The polarizations (P) obtained upon stimulation with nothing (P-0), encephalitogenic factor (P-EF), phytohaemagglutinin (P-PHA), or melanoma antigen (P-MEL), and the ratios RR(ef) (P-EF/P-PHA) and RR(mel) (P-MEL/P-PHA) were lower for SCM-responding lymphocytes from the patients as a group than for those of the controls. The specificity and sensitivity of the IFFP tests (using cutoff values) to detect melanoma were 90.6 and 73.9%, respectively. The IFFP tests may facilitate the discrimination between melanoma patients and healthy subjects, and may be used in follow-up of patients with melanoma.