Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1F0-type ATPase

Mutants of ECF1-ATPase were generated, containing cysteine residues in one or more of the following positions: alphaSer-411, betaGlu-381, and epsilonSer-108, after which disulfide bridges could be created by CuCl2 induced oxidation in high yield between alpha and epsilon, beta and epsilon, alpha and gamma, beta and gamma (endogenous Cys-87), and alpha and beta. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in epsilonSer-108 along with either the DELSEED region of beta (Glu-381) or the homologous region in alpha (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the epsilon subunit. In betaE381C/epsilonS108C the beta-epsilon cross-link was obtained preferentially when Mg2+ and ADP + Pi (addition of MgCl2 + ATP) was present, while the alpha-epsilon cross-link product was strongly favored in the alphaS411C/epsilonS108C mutant in the Mg2+ ATP state (addition of MgCl2 + 5'-adenylyl-beta,gamma-imidodiphosphate). In the triple mutant alphaS411C/betaE381C/epsilonS108C, the epsilon subunit bound to the beta subunit in Mg2+-ADP and to the alpha subunit in Mg2+-ATP, indicating a significant movement of this subunit. The gamma subunit cross-linked to the beta subunit in higher yield in Mg2+-ATP than in Mg2+-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the beta over the alpha subunit.     

Comparative study on adaptive rational macromodels of highly resonant structures

Two macromodeling techniques using rational bases are investigated to accurately predict the natural frequencies of highly resonant microwave structures. Three methods are proposed and compared to calculate pole‐free solutions to the Thiele continued fraction and vector fitting pole‐residue models of the characteristic equation det[Z(s)]. © 2007 Wiley Periodicals, Inc. Int J RF and Microwave CAE, 2007.     

Intramuscular collagen characteristics of ram, wether, and zeranol-implanted ram lambs

Eighteen spring-born Columbia ram, wether, and zeranol-implanted ram lambs were studied to determine the influence of castration or zeranol implants on intramuscular collagen (IMC) properties and muscle shear force values. Warner-Bratzler shear force values for longissimus muscle were greatest for ram lambs, intermediate for implanted rams, and least for wethers (P < .05). Nonreducible collagen crosslink concentration was greater in IMC of rams and implanted rams (P < .05). The IMC from rams compared with that from wethers contained proportionately more Type III than Type I collagen (P < .05); values for implanted rams were intermediate. Heat-soluble muscle collagen concentration was greater for rams and implanted rams than for wethers (P < .05); however, insoluble collagen concentration did not differ by treatment. Muscle collagen concentrations were not different for rams, wethers, or implanted rams. Increased shear force values in rams were associated with elevated collagen crosslink concentration and increased proportion of Type III collagen. Greater concentration of soluble collagen in ram IMC neither diminished nor diluted IMC crosslinking. The proportion of heat-labile collagen in the fractions did not reflect the IMC crosslinking profile for ram and wether lambs. Zeranol implantation modified IMC characteristics of rams such that shear force values and some collagen properties were similar to those of wethers.     

Vorticity field structure associated with the 3D Tollmien-Schlichting waves

Details of the vorticity field structure associated with the 3D Tollmien-Schlichting waves have been examined based upon the recent numerical studies of the subject. First, a single obliquet-s wave has been found to have the velocity component parallel to the wave front playing an overall dominant role, in particular, to create the longitudinal vorticity. The so-called Benney-Lin longitudinal vortices are then demonstrated to be, in fact, a minor consequence compared with the localized longitudinal vorticity field and its periodic pumping. Finally, the formation of the longitudinal vorticity field in the fundamental- and subharmonic-mode interactions is explained. The research reported in this paper has been supported in part by Bundesministerium für Forschung und Technologie and by Deutsche Forschungsgemeinschaft. The major part of the paper has been presented at the Third Asian Congress of Fluid Mechanics, 1–5 September 1986, in Tokyo, as a General Lecture by the senior author, FRH.     

Presence of X-prolyl-dipeptidyl-peptidase in lactic acid bacteria

Prolyl-dipeptidyl-peptidase activity was detected in cell extracts of 21 lactic acid bacteria tested. Using disc electrophoresis and various substrates it was possible to distinguish it from proline iminopeptidase and proline endopeptidase. Generally the activity was high and was greater than that of proline iminopeptidase of proline endopeptidase at neutral pH and at 25 degrees C.     

Implementing nurse sensitive outcomes into care planning at a long-term care facility

This article describes one long-term care facility's efforts to implement standardized language in the care planning process. Federal regulations for long-term care mandate the use of a uniform comprehensive assessment tool. Eighteen Resident Assessment Protocols (RAPs) are identified for data collection. Computer databases were revised for care planning. Appropriate North American Nursing Diagnosis Association (NANDA) diagnoses were linked to each RAP. Nursing-Sensitive Outcomes (NOCs) were linked to each NANDA as goals. Nursing Interventions Classifications (NICs) were linked to NANDA diagnosis and NOC outcomes as approaches. The databases are illustrated, and frequently used NANDAs and NOCs are identified.     

Culture and characterization of sinusoidal endothelial cells isolated from human liver

BACKGROUND: Epidemiologic data concerning skin diseases in many rural areas in sub-Saharan Africa are not available. Little is known about the effect of regular treatment schedules by paramedical staff (especially community health workers) in the primary healthcare system on the severity and prevalence of dermatoses. METHODS: 5780 school and pre-school children from 13 primary schools in four sublocations in rural western Kenya (Kisumu District) were examined for dermatoses by the author, together with community health workers in 1993. On-the-spot training and weekend seminars about important and common dermatoses were also given. In 1994 a dermatology program was started within the primary healthcare system. Twelve trained community health workers carried out regular school visits once a week and diagnosed and treated pupils with dermatoses. Treatment was performed with gentian violet 1% solution for bacterial skin infections, Whitfield's ointment for dermatophytoses, benzylbenzoate emulsion 25% for scabies, and hydrocortisone acetate 1% cream for eczemas. All schools were visited again in 1995 to evaluate the long-term effects of the program. RESULTS: In 1993, the prevalence rate for dermatoses was 32.4%. Most of the skin diseases found were of infective origin (27.1% were caused by bacteria, 21.6% by fungi, and 17.6% by arthropods, mainly scabies mites). Dermatitis accounted for 3.5%. In 1995, the prevalence of dermatoses declined to 29.6% (p<0.05), and this reduction was most strongly observed for tropical ulcers and tinea capitis. Additionally, there was an improvement in the extent and severity of skin diseases. CONCLUSIONS: This study defines, for the first time, the number and extent of skin diseases in children in rural Kisumu District; most dermatoses were of infective origin. The study demonstrates that community health workers in the primary healthcare system are capable of dealing successfully with the most common dermatoses in children following a short training period.     

Investigation of I1-imidazoline receptors using microphysiometry and molecular modelling

A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.     

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.     

Wave-front curvature as a cause of slow conduction and block in isolated cardiac muscle

We have investigated the role of wave-front curvature on propagation by following the wave front that was diffracted through a narrow isthmus created in a two-dimensional ionic model (Luo-Rudy) of ventricular muscle and in a thin (0.5-mm) sheet of sheep ventricular epicardial muscle. The electrical activity in the experimental preparations was imaged by using a high-resolution video camera that monitored the changes in fluorescence of the potentiometric dye di-4-ANEPPS on the surface of the tissue. Isthmuses were created both parallel and perpendicular to the fiber orientation. In both numerical and biological experiments, when a planar wave front reached the isthmus, it was diffracted to an elliptical wave front whose pronounced curvature was very similar to that of a wave front initiated by point stimulation. In addition, the velocity of propagation was reduced in relation to that of the original planar wave. Furthermore, as shown by the numerical results, wave-front curvature changed as a function of the distance from the isthmus. Such changes in local curvature were accompanied by corresponding changes in velocity of propagation. In the model, the critical isthmus width was 200 microns for longitudinal propagation and 600 microns for transverse propagation of a single planar wave initiated proximal to the isthmus. In the experiments, propagation depended on the width of the isthmus for a fixed stimulation frequency. Propagation through an isthmus of fixed width was rate dependent both along and across fibers. Thus, the critical isthmus width for propagation was estimated in both directions for different frequencies of stimulation. In the longitudinal direction, for cycle lengths between 200 and 500 milliseconds, the critical width was < 1 mm; for 150 milliseconds, it was estimated to be between 1.3 and 2 mm; and for the maximum frequency of stimulation (117 +/- 15 milliseconds), it was > 2.5 mm. In the transverse direction, critical width was between 1.78 and 2.32 mm for a basic cycle length of 200 milliseconds. It increased to values between 2.46 and 3.53 mm for a basic cycle length of 150 milliseconds. The overall results demonstrate that the curvature of the wave front plays an important role in propagation in two-dimensional cardiac muscle and that changes in curvature may cause slow conduction or block.