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.     

Indocyanine green: intracellular uptake and phototherapeutic effects in vitro

Indocyanine green (ICG; absorption peak in human plasma 805 nm) was investigated for ICG-mediated phototherapy in vitro. The cellular uptake of ICG (1 microM-50 microM) into HaCaT keratinocytes after an incubation period of 24 h increased up to an intracellular ICG concentration of 12.1 +/- 1.3 nmol per 10(6) cells. To examine dose dependent phototoxic effects in vitro, keratinocytes were incubated with 0 microM-50 microM ICG for 24 h and irradiated by a diode laser (805 nm) with different energy densities (0, 12, 24, 48 J cm-2). All applied ICG concentrations except for 5 microM yielded a cell killing effect in combination with irradiation depending significantly on ICG concentration and light dose. Cell viability for dark control and cells incubated with 50 microM ICG and irradiated with 48 J cm-2 was 0.82 +/- 0.15 and 0.07 +/- 0.02, respectively. Sodium azide (100 mM), a quencher of reactive oxygen species, inhibited significantly the cell killing using 50 microM ICG and 24 J cm-2. Taken together, photoactivation of ICG by irradiation with a diode laser was shown to induce effectively cell killing of HaCaT keratinocytes. Moreover, this effect was inhibited by sodium azide, thus irradiation of ICG might induce a photodynamic reaction.     

Spontaneous bacterial peritonitis in patients with ventriculoperitoneal shunts

A questionnaire to evaluate the needs of dialytic patients and the burden that these have on the health service and staff, was formulated by the nurses. The results showed how the patient's case history indicate, significantly, not only personal and subjective aspects (e.g. interpersonal relationships), but also how they construct their relationships with the hospital and its service (including the hospital staff). This opens up certain reflections on what role communication and information play within the hospital structure.     

The L5178Y/tk+/- mouse lymphoma specific gene and chromosomal mutation assay a phase III report of the U.S. Environmental Protection Agency Gene-Tox Program

The L5178Y/tk+/- (-)3.7.2C mouse lymphoma assay (MLA) which detects mutations affecting the heterozygous thymidine kinase (tk) locus is capable of responding to chemicals acting as clastogens as well as point mutagens. Improvements in the assay to enhance detection of this spectrum of genetic events are summarized, and criteria for evaluating the data are defined. Using these criteria, the Phase III Work Group reviewed and evaluated literature containing MLA results published from 1976 through 1993. The data base included 602 chemicals of which 343 were evaluated as positive, 44 negative, 18 equivocal, 54 apparently inappropriate for evaluation in this test system with the published protocols, and 142 that were inadequately tested, and thus a definitive call could not be made. The overall performance of the assay is summarized by chemical class, and the outcome of testing 260 chemicals in the MLA is compared with Gene-Tox and National Toxicology Program evaluations of rodent carcinogenesis bioassay results for the same chemicals. Based on the Work Group's evaluation of published MLA data for chemicals that were considered adequately tested, it is concluded that for most chemicals the L5178Y/tk+/- mouse lymphoma assay is eminently well suited for genotoxicity testing and for predicting the potential for carcinogenicity.     

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.