Development and validation of transient isotachophoretic capillary zone electrophoresis for determination of peptides

Although capillary zone electrophoresis (CZE) is known for its high resolution power and low mass detection limits, the concentration detection limits are rather poor when ultraviolet absorbance detection is used. To overcome this limitation, several on-column transient isotachophoresis (tITP) protocols have been developed and validated for the determination of both cationic and anionic model peptides, separately. Using this preconcentration method, up to 72% of the capillary can be filled with sample solution, without any loss in resolution. Thus, without any modification of the hardware set-up, the sensitivity is increased about two orders of magnitude. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility is observed in the 20 to 100 ng/mL concentration range. For the anionic peptides (N-t-Boc-Pentagastrin and two related peptides), a tITP method was developed using a dynamically coated capillary. The coating was prepared by adding Fluorad FC-135 to the leading electrolyte buffer. In this way a positively charged bilayer was formed on the inside of the capillary, producing an electroosmotic flow towards the outlet using reversed polarity conditions. In this way, acceptable analysis times were achieved. Using the developed tITP method, up to 72% of the capillary can be filled with sample solution as well. The anionic peptides are separated even better than when using CZE conditions. Linearity and reproducibility in the 20-100 ng/mL range proved to be excellent.     

Nonpeptide oxytocin antagonists: potent, orally bioavailable analogs of L-371,257 containing a 1-R-(pyridyl)ethyl ether terminus

Structure-activity studies on the oxytocin antagonist 1 (L-371,257) have identified a new series of high affinity, receptor-selective OT antagonists in which the N-acetyl-4-piperidinyl ether terminus in 1 has been replaced with a 1-(aryl)ethoxy group.     

Prognostic factors in surgically treated small cell cervical carcinoma followed by adjuvant chemotherapy

BACKGROUND: Small cell carcinoma of the uterine cervix is an uncommon tumor associated with high mortality even among patients with early stage disease. The role of adjuvant chemotherapy after surgery has been suggested by regimens used for small cell lung carcinoma. During the years 1980-1997, 19 cases in which various adjuvant chemotherapies were given after hysterectomy were reported in the literature published in English. METHODS: Adjuvant chemotherapy was administered consecutively to 23 patients with Stage Ib-II small cell cervical carcinoma who had been primarily treated with radical hysterectomy and had adequate bone marrow, renal, and hepatic functions. A combination of vincristine, doxorubicin, and cyclophosphamide alternating with cisplatin and etoposide (VAC/PE) was administered to 14 patients during the years 1988-1996 according to a prospective study protocol. A combination of cisplatin, vinblastine, and bleomycin (PVB) was administered to 8 patients, and another regimen was administered to 1 patient during the years 1984-1988. Prognostic factors were evaluated by analyzing both the data on these 23 patients and the pooled data on the cases retrieved from the literature and our own files. RESULTS: Ten of the 14 patients who received VAC/PE had no evidence of disease during a median follow-up of 41 months, whereas 3 of the 9 who received PVB or another regimen survived. Of the 10 patients who died of their disease, all died of distant metastasis within 10 months after recurrence. Meta-analysis of the pooled data showed that 68% of patients who received regimens containing VAC or PE survived, whereas 33% of patients who received regimens not containing VAC/PE survived (P = 0.0078, log rank test). Seventy percent of patients with no lymph node metastasis at hysterectomy and 35% with lymph node metastasis survived (P = 0.05). All patients who died of disease had extrapelvic metastasis. CONCLUSIONS: Chemotherapies containing VAC or PE are favorable regimens for patients with early stage small cell cervical carcinoma after radical hysterectomy.     

Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding

The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy. Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms). These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves. The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol). Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface. This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure.     

Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.     

Cyclosporin A and hydroxycyclosporine (M-17) affect the secretory phenotype of human gingival fibroblasts

The responsiveness of human gingival fibroblast populations to cyclosporin A (CsA) and its principal metabolite, hydroxycyclosporine (M17), was evaluated in cell culture. Gingival fibroblasts exhibited a dose-dependent accumulation and bell-shaped distribution of dansylated CsA. A 100-fold excess of non-labeled CsA prevented the accumulation of the fluorescent probe in the fibroblasts. Both CsA (400 ng/ml) and M17 (100 ng/ml) stimulated mean gingival fibroblast cell number to 23.2% and 36.7% above controls, and reduced mean collagen production by 37.7% and 37.4% below controls, respectively; however, neither CsA nor M17 affected mean protein production in comparison to control cultures. Analyses of responses to CsA and M17 by ligand-accumulating and non-accumulating fibroblasts sorted out from the parent cultures did not provide consistent interstrain responses either by cells representing the upper quartile of fluorescence or cells representing the bottom quartiles of fluorescence. These data demonstrate that CsA is accumulated by gingival fibroblasts and that CsA and M17 are potent modulators of gingival fibroblast phenotype.     

Fruit and vegetable consumption, nutritional knowledge and beliefs in mothers and children

Increasing fruit and vegetable consumption is an important health behaviour. Parental and other psychosocial influences on children's fruit and vegetable consumption are poorly understood. The contribution of a variety of psychosocial and environmental factors to consumption of fruit and vegetables by children aged 9-11 years was explored. Ninety-two mothers and children (48 girls and 44 boys) were recruited via urban primary health-care practices. Socio-economic and educational level, nutritional knowledge and health- and diet-related beliefs and attitudes were assessed in mothers and children by questionnaires and semistructured interviews. Mothers> diets were measured by a food frequency questionnaire, while children's diets were assessed by 3-day diaries (N=80). The pattern of influence of the various measures on fruit and vegetable consumption was compared with that on children's confectionery intake. The children's intakes of macronutrients were typical for the U.K. (37% fat, 50% carbohydrate and 13% protein by energy; 12 g/day fibre), while median fruit, fruit juice and vegetable intake amounted to about 2.5 servings/day. Univariate correlations and subsequent multiple regression analyses revealed quite different influences on the three food types. Independent predictors of children's fruit intake included mothers> nutritional knowledge (beta=0.37), mothers> frequency of fruit consumption (beta=0.30) and mothers> attitudinal conviction that increasing fruit and vegetable consumption by their children could reduce their risk of developing cancer (beta=0.27; multiple r2=0.37,p<0.0001). Children's vegetable consumption was independently explained by the child's liking for commonly eaten vegetables (beta=0.36) and the mother's belief in the importance of disease prevention when choosing her child's food (beta=-0.27 r2=0.20,p<0.001). Children's confectionery consumption was predicted by the mother's liking for confectionery (beta=0.32) and the children's concern for health in choosing what to eat (beta=-0.26 r2=0.16, p<0.005). Children's consumption of fruit and vegetables are related to different psychosocial and environmental factors. Promotion of this behaviour may require attention to nutritional education and child feeding strategies of parents.     

Long-term detection of microchimaerism in peripheral blood after pretransplantation blood transfusion

Renal allograft survival is prolonged after pretransplantation blood transfusion. The aim of this study was to test retrospectively the development and persistence of microchimaerism after pretransplantation blood transfusion and to assess whether the type of blood transfusion (partially matched [= sharing of at least one HLA-B and one HLA-DR antigen between blood donor and recipient] versus mismatched) influences the (continued) presence of donor-type cells. A sensitive nested PCR technique based on HLA-DRB1 allele-specific amplification using sequence-specific primers (detection level: one donor cell among 10(5) recipient cells) for detection of donor cells was implemented in our laboratory. We studied 21 patients for microchimaerism in the peripheral blood compartment, following blood transfusion. Our preliminary data show that microchimaerism was detectable up to 8 weeks after blood transfusion. In all patients receiving a partially matched blood transfusion, donor-type cells were detected in the first week after transfusion, in 7/8 patients 2-4 weeks after transfusion, and in some patients up to 8 weeks after transfusion. After mismatched transfusion a tendency to shorter duration of microchimaerism was observed.     

Complete MR angiography and Doppler ultrasound as the sole imaging modalities prior to carotid endarterectomy

OBJECTIVE: To assess the combination of duplex Doppler ultrasound (DUS) and complete carotid magnetic resonance angiography (MRA) for the non-invasive imaging of carotid disease and their effect on outcomes. Determine inter-reader agreement of carotid MRA. MATERIALS AND METHODS: One-hundred and ten carotid bifurcations were evaluated using DUS, 2D and 3D time-of-flight MRA from the aortic arch to the Circle of Willis in 55 patients. Percentage stenoses were determined by two blinded readers using standardized criteria. Clinical follow-up was by chart review. RESULTS: Correlation of Doppler and MRA was excellent (r=0.903, P<0.001). Inter-reader agreement (K) for MRA was good: internal carotid artery (ICA) (0.750), external carotid artery (ECA) (0.674) and common carotid artery (CCA) (0.410). Differences in CCA readings were due to minor differences in categorizing lesions as CCA versus ICA or ECA. MRA and Doppler detected nine occluded ICAs. Two DUS occlusions had ICA flow by MRA; one due to a reconstituted precavernous ICA, one a near occluded vessel. Five patients (9%) had surgical management modified by MRA with four not having surgery: three distal ICA/Siphon occlusions and one less severe stenosis by MRA. One tandem lesion not visualized by DUS was surgically significant. Nine aortic arch abnormalities had no surgical impact, possibly due to small sample size. Of 41 endarterectomies, there were no complications from errors of diagnosis. CONCLUSION: Carotid MRA correlates well with DUS with good inter-reader agreement. MRA confirms Doppler findings, expands anatomical information and identifies tandem lesions from the aortic arch to the Circle of Willis which can affect surgical management. This approach to carotid artery imaging appears to have no negative effect on surgical outcome.