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.     

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.     

A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides

During the last decade, episodes of sepsis have increased and Escherichia coli has remained the most frequent clinical isolate. Lipopolysaccharides (LPS; endotoxin) are the major toxic and antigenic components of gram-negative bacteria and qualify as targets for therapeutic interventions. Molecules that neutralize the toxic effects of LPS are actively investigated. In this paper, we describe a murine monoclonal antibody (MAb; WN1 222-5), broadly cross-reactive and cross-protective for smooth (S)-form and rough (R)-form LPS. As shown in enzyme-linked immunosorbent assay and the passive hemolysis assay, WN1 222-5 binds to the five known E. coli core chemotypes, to Salmonella core, and to S-form LPS having these core structures. In immunoblots, it is shown to react with both the nonsubstituted core LPS and with LPS carrying O-side chains, indicating the exposure of the epitope in both S-form and R-form LPS. This MAb of the immunoglobulin G2a class is not lipid A reactive but binds to E. coli J5, an RcP+ mutant which carries an inner core structure common to many members of the family Enterobacteriaceae. Phosphate groups present in the inner core contribute to the epitope but are not essential for the binding of WN1 222-5 to complete core LPS. Cross-reactivity for clinical bacterial isolates is broad. WN1 222-5 binds to all E. coli clinical isolates tested so far (79 blood isolates, 80 urinary isolates, and 21 fecal isolates) and to some Citrobacter, Enterobacter, and Klebsiella isolates. This pattern of reactivity indicates that its binding epitope is widespread among members of the Enterobacteriaceae. WN1 222-5 exhibits biologically relevant activities. In vitro, it inhibits the Limulus amoebocyte lysate assay activity of S-form and R-form LPS in a dose-dependent manner and it neutralizes the LPS-induced release of clinically relevant monokines (interleukin 6 and tumor necrosis factor). In vivo, WN1 222-5 blocks endotoxin-induced pyrogenicity in rabbits and lethality in galactosamine-sensitized mice. The discovery of WN1 222-5 settles the long-lasting controversy over the existence of anti-core LPS MAbs with both cross-reactive and cross-protective activity, opening new possibilities for the immunotherapy of sepsis caused by gram-negative bacteria.     

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.     

Heterotopic replantation

In multidigital amputations, it is sometimes better to replant an amputated finger to a different proximal part if a better function can be expected in this position. In our clinical material between October 1991 and March 1994, heterotopic replantation was performed in twelve digits in eleven patients. Three fingers were replanted to the thumb, three to the index, four to the middle, and two to the ring fingers. The functional results were satisfying. The total active range of motion was on an average 24% of a normal finger. Static two-point discrimination was 8.5 mm on an average, and the values for the Semmes-Weinstein test ranged between 3.61 and 6.5. A heterotopic replantation is of special value for primary thumb reconstruction.     

ALUM SHALE BITUMEN MATURATION AND MIGRATION: IMPLICATIONS FOR GOTLAND'S OIL

Thermal maturation of Swedish Alum Shale kerogen and bitumen has been determined from core samples from Eastern and Central Sweden. In samples from Eastern Sweden (Öland and Gotland), the kerogen and bitumen are thermally immature with respect to petroleum generation. In some areas of Central Sweden (Närke, ÖstergÖtland, and Kinnekulle in VästergÖtland), the kerogen is immature, whereas the bitumen is marginally mature to mature. In other areas of Central Sweden (Halleberg-Hunneberg in VästergÖtland), the kerogen is supermature and the bitumen mature. This suggests that bitumen from a mature source-rock has migrated into the Alum Shales of Central Sweden. Migration in Central Sweden is further evidenced by the occurrence of obviously-migrated bituments in vugs and voids in the organicpoor Ordovician limestone overlying the Alum Shale in Central Sweden, and in concretions within the Alum Shale itself. Based on biomarker distributions of extracted bitumen, Alum Shale kerogen pyrolysate and obviously-migrated oils, and the fact that the Alum Shale in most of the areas studied is the only petroleum source-rock extant, it is suggested that the migrated bitumen in Central Sweden is from the Alum Shale itself. Bitumen has migrated from areas where the Alum Shale is in close proxmity to Permo-Carboniferous intrusions, such as Halleberg-Hunneberg, into nearby areas such as Närke and ÖstergÖtland, where there is no evidence of intrusion and the indigenous organic matter is thermally immature. Other areas, where Alum Shales were associated with intrusions and consequently sourced oil, may have been eroded away. There are producing wells on the island of Gotland, where the Alum Shale is also thermally immature. It is therefore assumed that heating which was responsible for generating Gotland's oil was very localized (such as by an intrusion) or that the oil has migrated from a thermally moremature, distant area. On the basis of reservoir rock porosity, and the fact that the Alum Shale of Gotland contains no migrated component, localized heating is favored.     

The boundary contour method for two-dimensional Stokes flow and incompressible elastic materials

 A variant of the boundary element method, called the boundary contour method (BCM), offers a further reduction in dimensionality. Consequently, boundary contour analysis of two-dimensional (2-D) problems does not require any numerical integration at all. While the method has enjoyed many successful applications in linear elasticity, the above advantage has not been exploited for Stokes flow problems and incompressible media. In order to extend the BCM to these materials, this paper presents a development of the method based on the equations of Stokes flow and its 2-D kernel tensors. Potential functions are derived for quadratic boundary elements. Numerical solutions for some well-known examples are compared with the analytical ones to validate the development. Received 28 August 2001 / Accepted 15 January 2002