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This article outlines the problems of interprofessional practice in the hospital setting. It examines the difficulties associated with competing ideologies and aims, inequalities in power relations, communication and role confusion and overlap. Within the hospital setting such problems often arise although the roots of the problem may go unacknowledged. The ways in which the problems occur in the hospital setting are considered, as is the underlying cause of such problems. Case studies will be used to analyse how the problems arise and ways in which they might be addressed.

In exploratory scale biotechnological process development, the product must be rapidly identified although a reference compound may not always be available. LC-diode-array detection and MS were used for this purpose in a process producing 9alpha-hydroxyprogesterone from progesterone as substrate. The electrospray ionization mass spectrometer was combined with an ion trap mass spectrometer for the second generation MS. The preliminary identification, which could be carried out within the course of a day, confirmed that the product was a hydroxyprogesterone. The final identification step, which was much more material intensive and hence time consuming, involved a two-step preparative separation to yield quantities necessary for definitive product identification based on 1H- and 13C NMR.

This paper reports on the evaluation stage of an action research project on interprofessional collaboration in discharge planning. Findings from interviews with health care professionals working in the acute sector had revealed concerns about discharge planning and multidisciplinary teamwork. In the United Kingdom the National Health Service (NHS) Plan has reinforced the need for an integrated approach to health care. Effective health care integration requires effective communication, teamwork and the commitment to deliver integrated care. Integrated documentation is a key strategy for enhancing interprofessional collaboration and reducing the isolation of professionals, and has been successfully implemented in a range of health care settings. Presented with the concerns about the collaborative process in discharge planning, an action research strategy was chosen to bring about change in an orthopaedic ward in one London teaching hospital. This paper will evaluate the implementation of an integrated care pathway with fractured neck of femurs in one London teaching hospital. Care pathways facilitate the management of defined patient groups using interdisciplinary plans of care. The emphasis will be on understanding whether integrated care pathways enhance and develop interprofessional collaboration and enable effective information access and flow across the professions and the organization. The criteria for evaluation, forming the hypotheses of the study, were that interprofessional nonverbal and verbal communication would be enhanced and that interprofesisonal collaboration would increase. Methods of evaluation used were: (i) stakeholder interviews, (ii) interprofessional audit and (iii) analysis of the variances from the integrated care pathway. The evaluation revealed that although integrated care pathways led to improved outcomes for the health care trust there was little evidence to suggest that interprofessional relationships and communication were enhanced. Furthermore, key factors in discharge delays appeared to be organizational rather than professional.

This study reports on the effect of home-based mental health treatment following psychiatric hospitalization on ambulatory follow-up rates and readmission rates in a Medicaid managed care population. Logistic regression models were used to predict the odds of ambulatory treatment after hospitalization and to predict rehospitalization. A consumer who received in-home treatment was 22 times more likely to follow-through with aftercare treatment of more than one visit than were those who did not receive in-home treatment. However, in-home treatment, age, gender, and previous hospitalization did not significantly predict the odds of rehospitalization. While home-based services did not reduce the incidence of rehospitalization, providing services within the home has the potential to increase attendance in aftercare services by Medicaid managed care recipients.

We investigated a novel strategy for measuring the synthesis rate of proteins in skeletal and cardiac muscle. Mass isotopomer distribution analysis allows measurement of the isotopic enrichment of the true biosynthetic precursor for proteins (tRNA-amino acids), but cannot easily be applied to slow turnover muscle proteins due to insufficient isotope incorporation into multiply labeled species. Using a rapid turnover protein from the same tissue, however, might reveal tRNA-amino acid enrichment. We tested this strategy in rats on muscle creatine kinase (CK). A trypsinization peptide (3647u) containing 5 leucine repeats was identified by computer-simulated digestion of CK and then isolated from trypsin hydrolysates. Mass isotopomer abundances were determined by electrospray ionization-magnetic sector-mass spectrometry after in vivo administration of [(2)H(3)]leucine. Myosin heavy chain was also isolated and hydrolyzed to free amino acids. Muscle tRNA-amino acids were well labeled, by direct measurement. Enrichments of M(+1) and M(+2) mass isotopomers in the CK-peptide were measurable but low (consistent with a CK half-life of 3-10 days). Incorporation into skeletal muscle myosin indicated a half-life of 54 days. In conclusion, the general strategy of measuring protein kinetics by quantifying mass isotopomer abundances of mid-sized peptides from protein hydrolysates is effective, but CK does not turn over rapidly in muscle, contrary to previous reports. Identification of a rapid turnover muscle protein would be useful for this purpose.

The nudF and nudC genes of the fungus Aspergillus nidulans encode proteins that are members of two evolutionarily conserved families. In A. nidulans these proteins mediate nuclear migration along the hyphae. The human ortholog of nudF is Lis1, a gene essential for neuronal migration in the developing cerebral cortex. The mammalian ortholog of nudC encodes a protein that interacts with Lis1. We have identified orthologs of nudC and Lis1 from the nematode Caenorhabditis elegans. Heterologous expression of the C. elegans nudC ortholog, nud-1, complements the A. nidulans nudC3 mutant, demonstrating evolutionary conservation of function. A C. elegans nud-1::GFP fusion produces sustained fluorescence in sensory neurons and embryos, and transient fluorescence in the gonad, gut, vulva, ventral cord, and hypodermal seam cells. Fusion of GFP to C. elegans lis-1 revealed expression in all major neuronal processes of the animal as well as the multinucleate spermathecal valves and adult seam cells. Phenotypic analysis of either nud-1 and lis-1 by RNA interference yielded similar phenotypes, including embryonic lethality, sterility, altered vulval morphology, and uncoordinated movement. Digital time-lapse video microscopy was used to determine that RNAi-treated embryos exhibited nuclear positioning defects in early embryonic cell division similar to those reported for dynein/dynactin depletion. These results demonstrate that the LIS-1/NUDC-like proteins of C. elegans represent a link between nuclear positioning, cell division, and neuronal function.

We investigated the effects of one-trial fear conditioning on phospholipase C-beta1a catalytic activity and protein level in hippocampal formation and medial frontal cortex of untreated control rats and rats prenatally exposed to ethanol. One hour following fear conditioning of untreated control rats, phospholipase C-beta1a protein level was increased in the hippocampal cytosolic fraction and decreased in the hippocampal membrane and cortical cytosolic and cortical membrane fractions. Twenty-four hours after fear conditioning, phospholipase C-beta1a protein level was reduced in the hippocampal cytosolic fraction and elevated in the cortical nuclear fraction; in addition, 24 h after conditioning, phospholipase C-beta1a activity in the cortical cytosolic fraction was increased. Rats that were exposed prenatally to ethanol displayed attenuated contextual fear conditioning, whereas conditioning to the acoustic-conditioned stimulus was not different from controls. In behavioral control (unconditioned) rats, fetal ethanol exposure was associated with reduced phospholipase C-beta1a enzyme activity in the hippocampal nuclear, cortical cytosolic, and cortical membrane fractions and increased phospholipase C-beta1a protein level in the hippocampal membrane and cortical cytosolic fractions. In certain cases, prenatal ethanol exposure modified the relationship between fear conditioning and changes in phospholipase C-beta1a protein level and/or activity. The majority of these effects occurred 1 h, rather than 24 h, after fear conditioning. Multivariate analysis of variance revealed interactions between fear conditioning, subcellular fraction, and prenatal ethanol exposure for measures of phospholipase C-beta1a protein level in hippocampal formation and phospholipase C-beta1a enzyme activity in medial frontal cortex. In the majority of cases, fear conditioning-induced changes in hippocampal phospholipase C-beta1a protein level were augmented in rats prenatally exposed to ethanol. In contrast, fear conditioning-induced changes in cortical phospholipase C-beta1a activity were, often, in opposite directions in prenatal ethanol-exposed compared to diet control rats. We speculate that alterations in subcellular phospholipase C-beta1a catalytic activity and protein level contribute to contextual fear conditioning and that learning deficits observed in rats exposed prenatally to ethanol result, in part, from dysfunctions in phospholipase C-beta1a signal transduction., (Copyright 2001 Academic Press.)

The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP shares significant sequence similarity with the protein and lipid 3-phosphatase myotubularin, and with several other members of the myotubularin gene family including SET-binding factor 1. However, unlike myotubularin, 3-PAP does not contain a consensus HCX(5)R catalytic motif. The 3-PAP sequence contains several motifs that predict interaction with proteins containing Src homology-2 (SH2) domains, phosphotyrosine-binding (PTB) domains, members of the 14-3-3 family, as well as proteins with SET domains. Northern blot analysis identified two transcripts (5.5 kb and 2.5 kb) with highest abundance in human liver, kidney, lung, and placenta. 3-PAP immunoprecipitates isolated from platelet cytosol hydrolyzed the D3-phosphate from PtdIns(3)P and PtdIns 3,4-bisphosphate [PtdIns(3,4)P(2)]. However, insect cell-expressed 3-PAP recombinant protein was catalytically inactive, confirming our prior prediction that this polypeptide represents an adapter subunit.

This work outlines the fundamental scaling laws associated with electrical field flow fractionation channels. Although general FFF theory indicates few advantages from miniaturization, EFFF theory indicates clear advantages to miniaturization of the EFFF channel. Retention, plate heights, resolution, equilibration times, and time constants are examined. The outlined theory predicts scaling advantages in each of these areas after miniaturization. Potential applications, such as the use of these systems for sample preparation in microscale total analysis systems, and improvements associated with these theoretical predictions are also discussed.

Nurse education has been subject to many changes and much debate and criticism over recent years. What has become increasingly evident is that with the changing nature of nursing within society, nursing curricula have to be more flexible and dynamic if they are to meet a multiplicity of needs. There is also a need to recognize that many levels of curricula will be required to prepare the nurses of the future. At Middlesex University the development of specialist practice programmes at postgraduate diploma level, and preparation of nurses for a higher level of practice at masters level has required the development of a new curriculum model which allows both the individualization of academic programmes to meet the needs of nurses, their clients and the organization in which they work, and the integration of development and learning through practice. This model is built on the results of an evaluation of an existing postgraduate programme in interprofessional health care. Key features of the curriculum development include a structured collaboration between student, practice mentor and academic supervisor, and the use of a professional development portfolio to individualize the academic programme and facilitate autonomous learning., (Copyright 2001 Harcourt Publishers Ltd.)

Most biomaterials can be rendered adhesive for anchorage-dependent cells by adsorption of serum, isolated extracellular matrix proteins, or immobilization of peptide sequences. However, difficulties are frequently encountered in characterizing the adsorbed layer due to conformational changes in the molecules following adsorption and interference from nonspecifically adsorbed molecules. In this study, we have investigated a technique for covalently immobilizing fibronectin to the PEO-containing triblock copolymer Pluronic F108 ("F108"). We have compared this technique to solution adsorption of fibronectin for its ability to provide controlled variation of bound fibronectin and regulation of fibroblast behavior. Both simple adsorption and covalent immobilization were effective for varying substrate-bound fibronectin. However, adsorption of fibronectin did not effectively regulate fibroblast attachment or spreading in either serum-free or serum-containing media. Fibroblast attachment, spreading, cytoskeletal organization, and proliferation were effectively regulated in response to fibronectin immobilized to F108. Furthermore, F108-treated surfaces without immobilized fibronectin did not support nonspecific fibroblast attachment, even in the presence of serum-containing medium. Fibroblasts were observed to only proliferate on surfaces with high levels of immobilized fibronectin that supported extensive cell spreading and cytoskeletal organization. In summary, covalent immobilization of fibronectin to F108 provided controlled regulation of fibroblast behavior without interference from nonspecific protein adsorption, even in the presence of serum-containing medium.

The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.

Background: Toxic endothelial cell destruction (TECD) syndrome after intraocular ophthalmic surgery is rare and can result from exposure to a variety of toxins. During January 8 to 14, 1998, 6 patients developed TECD with corneal edema associated with unreactive or dilated pupils at Hospital A., Methods: A case patient was any Hospital A patient with TECD within 24 hours after surgery during January 5 to 14, 1998 (epidemic period). A control was any hospital A ophthalmic surgery patient without TECD during the epidemic period. The medical records of hospital A ophthalmology surgery patients during the pre-epidemic (ie, September 1, 1997-January 4, 1998) and epidemic periods were reviewed. Inductively coupled plasma atomic emission spectrometry was used to detect trace inorganic elements on sterilized surgical instruments. Cannulated surgical instruments and laboratory rinsates were perfused directly to the corneal endothelium of isolated rabbit and human corneas. Corneal endothelial ultrastructure and swelling were assessed., Results: The rate of TECD at hospital A was higher during the epidemic than pre-epidemic period (6/12 vs 0/118, P<.001). The only change during the periods was the introduction, on November 5, 1997, of a new sterilization method, AbTox Plazlyte, for sterilization of ophthalmic surgery instruments. Findings from spectrometry revealed that copper and zinc residues were higher in instruments sterilized with Plazlyte than in those sterilized with ethylene oxide (median copper value, 7.64 mg/L vs 0.14 mg/L, respectively, P =.02; median zinc value, 5.90 mg/L vs 1.35 mg/L, respectively, P =.2). Corneal endothelial perfusion of Plazlyte sterilized-instrument rinsates or laboratory solution with copper and zinc produced irreversible damage, similar to toxic corneal endothelial destruction, to rabbit and human corneas., Conclusion: A new sterilization method degraded brass to copper and zinc on cannulated surgical instruments resulting in TECD of the cornea. Arch Ophthalmol. 2000;118:1167-1176

Electrical field-flow fractionation (ElFFF) results for a series of polystyrene latex beads are presented. To first approximation, retention behavior can be related to conventional FFF theory, modified to account for a particle-wall repulsion effect. Size selectivity and column efficiency were exceptionally high, again approaching the upper limit predicted by theory. For the channel described in the present study, application of small voltages (typically less than 2 V) across the thin (131 microm) separation space defined by a Teflon spacer generates nominal field strengths of 10(4) V m(-1). However, electrode polarization reduces the effective field across the bulk of the channel to approximately 3% of the nominal value in the system studied. The magnitude of the applied field was calibrated by using standard latex beads of known size and mobility. Perturbations to retention behavior, such as overloading, were investigated. It was found that ideal separations occur at very dilute concentrations of the sample plug and that working in systems of very low ionic strength, the double-layer thickness adds significantly to the effective size of a particle. Steric inversion was observed at a particle size of approximately 0.4 microm under the conditions employed.

In order to understand the influence of cell-adhesive molecules on anchorage-dependent cell behavior on biomaterial surfaces, a model system is required where these molecules can be applied to surfaces with controlled surface ligand density and resistance to the adsorption of additional proteins present in the medium. This study asked whether fibronectin could be immobilized in a controlled manner to a hydrophobic surface with a chemically modified triblock surfactant. ELISA studies indicated that variation of the soluble fibronectin concentration used for immobilization could be used to control the amount of fibronectin immobilized to the surface. Furthermore, fibroblasts seeded on these surfaces in 10% serum-containing medium attached and spread as a function of the amount of immobilized fibronectin. Surfaces treated with unmodified surfactant did not support cell attachment, suggesting that cell attachment and spreading were primarily regulated by the immobilized fibronectin with minimal interference from adsorption of serum proteins. Together, these results suggest that covalent immobilization to Pluronic F108 provides a method for studying cellular responses to cell adhesive proteins with little interference from competing adsorbates, even in the presence of complex biological fluids such as serum. This technique may be applicable to a variety of existing hydrophobic biomedical polymers as a basic science tool as well as for influencing cell behavior at implant interfaces.

This work describes a method for coupling cell adhesion peptides to hydrophobic materials for the purpose of controlling surface peptide density while simultaneously preventing nonspecific protein adsorption. PEO/PPO/PEO triblock copolymers (Pluronic F108) were equipped with terminal pyridyl disulfide functionalities and used to tether RGD containing peptides to polystyrene (PS). The density of F108 on PS was 1.4 E5 +/- 2.12 E1 molecules/microm2. XPS and ToF SIMS indicated that the F108 coating was homogeneous and that the unmodified and activated F108 distributed evenly on PS. By mixing unmodified F108 with PDS-activated F108 prior to adsorption, it was possible to vary peptide density between 0 and 8.7 E4 +/- 2.66 E3 peptides/microm2, while otherwise, maintaining consistent surface properties. GRGDSY grafted PS supported cell attachment, spreading, and development of cytoskeletal structure, all of which were found to increase with increasing peptide density. Cell proliferation followed this same trend, however, maximal growth occurred at a submaximal peptide density. Cell aspect ratio varied in a biphasic manner with GRGDSY density. F108 coated PS and GRGESY grafted PS were inert to cell adhesion. Cells released from GRGDSY grafted PS upon addition of either a reducing agent or free GRGDSY, which indicates that cell-substrate interactions were mediated solely by the tethered peptides.

Purpose: Assessment of lower size limit of Sedimentation Field-Flow Fractionation (SedFFF), specifically to evaluate if the method is suitable to determine the size and size distribution of 20 nm colloidal gold particles with high resolution., Methods: Sedimentation Field-Flow Fractionation was used to determine the size of the colloidal particles. Due to the high density of gold it was possible to extend the lower size limit of SedFFF well below 20 nm. The size distribution of a gold colloid was obtained from the peak broadening caused by the polydispersity of the sample. The peak broadening due to instrumental imperfections was determined. For comparison purpose the particles were also sized using SEM and PCS., Results: The mean diameter of the particles was determined to be (20.87+/-0.05) nm, the standard deviation in size being 1.04 nm (about 5%). SEM could confirm that the particles are about 20 nm in diameter. A sizing with PCS was not possible. The particles have a strong tendency to aggregate and PCS yields a diameter that is much too large., Conclusions: At optimized analytical parameters Sedimentation Field-Flow Fractionation is an effective method to measure the size of gold particles as small as 15 nm with an accuracy of about 0.1 nm. The polydispersity of the sample can easily be determined.

A novel locus in the human Prader-Willi syndrome (PWS) region encodes the imprinted ZNF127 and antisense ZNF127AS genes. Here, we show that the mouse ZNF127 ortholog, Zfp127, encodes a homologous putative zinc-finger polypeptide, with a RING (C3HC4) and three C3H zinc-finger domains that suggest function as a ribonucleoprotein. By the use of RT-PCR across an in-frame hexamer tandem repeat and RNA from a Mus musculus x M.spretus F1interspecific cross, we show that Zfp127 is expressed only from the paternal allele in brain, heart and kidney. Similarly, Zfp127 is expressed in differentiated cells derived from androgenetic embryonic stem cells and normal embryos but not those from parthogenetic embryonic stem cells. We hypothesize that the gametic imprint may be set, at least in part, by the transcriptional activity of Zfp127 in pre- and post-meiotic male germ cells. Therefore, Zfp127 is a novel imprinted gene that may play a role in the imprinted phenotype of mouse models of PWS.

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production.

Objectives: There is a paucity of data describing the epidemiology of serious hearing impairment among children in the United States. This report provides information on the prevalence of serious hearing impairment among children born in the 1980s and living in the metropolitan Atlanta area in 1991-1993 and on the characteristics of children with serious hearing impairment., Methods: Data for this report are drawn from the Metropolitan Atlanta Developmental Disabilities Surveillance Program, an ongoing, active case-ascertainment system for mental retardation, cerebral palsy, hearing impairment, and vision impairment among children 3 to 10 years of age. Hearing impairment was defined as a bilateral, pure-tone hearing loss at frequencies of 500, 1000, and 2000 Hz averaging 40 decibels or more, unaided, in the better ear. Both severity and type of hearing loss were examined. Cross-sectional as well as birth cohort prevalence rates of serious hearing impairment were computed by sex and by race. The presence of mental retardation, cerebral palsy, vision impairment, or a seizure disorder was also assessed. An attempt was made to determine the probable etiology of a subset of the cases., Results: The average, annual prevalence rate for moderate to profound hearing loss was 1.1 per 1000. The prevalence rate increased steadily with age. Ninety percent of all cases for which the type of loss was recorded were sensorineural. The highest rate was seen among black male children (1.4 per 1000). Thirty percent of case children had another neurodevelopmental condition, most frequently mental retardation. Black male children also experienced the highest rate of presumed congenital hearing impairment. The mean age at which children with presumed congenital hearing impairment first met the surveillance case definition was 2. 9 years. A probable etiology could only be found for 22% of cases born in the study area., Conclusions: The data presented here provide information on the descriptive epidemiology of serious hearing impairment among United States children. The reasons for the higher rates among black children, especially males, may be a fruitful direction for further research.

The measurement of protein kinetics by isotopic techniques has been hindered by the long-standing difficulty of accurately measuring the isotope content of the biosynthetic precursor pool (aminoacyl-tRNA in tissues). Mass isotopomer distribution analysis (MIDA) is a stable isotope-mass spectrometric (MS) technique for measuring biosynthetic precursor enrichments from measurements on a polymeric product, based on combinatorial probabilities of labeled and unlabeled monomeric subunits. Proteins contain complex isotopomer patterns as a result of their relatively high molecular mass, however, so that resolution of the individual mass isotopomers in the polymeric product (an analytic requirement for MIDA) is technically difficult. An approach for measuring protein synthesis by MIDA is described and tested here: First, in vitro, using a synthetic peptide present in human serum albumin; and then, in vivo, for albumin synthetic rate in rats. A peptide contained in human serum albumin (SVVLLLR) and theoretically recoverable from trypsin/chymotrypsin proteolysis was synthesized by solid-phase peptide synthesis using known mixtures of natural abundance and [5,5, 5-2H3]leucine. Additionally, enriched and natural abundance peptides were mixed in vitro to simulate in vivo biosynthesis and to address problems of instrument accuracy, precision, and data management. The mass isotopomer patterns of the synthetic peptides were analyzed using electrospray ionization (ESI) with both magnetic sector and quadrupole mass analyzers. The resolution of the magnetic sector was superior to that of the quadrupole instrument, but accuracy and precision in the measurement of mass isotopomer abundances and kinetic parameters were comparable and both gave values close to those predicted. Next, rats were infused with [5,5,5-2H3]leucine intravenously, and a leucine-rich peptide was isolated and purified from trypsin-digested rat serum albumin (RHPDYSVSLLLR, 1456 Da) and then analyzed by ESI-MS using a magnetic sector instrument. Precursor pool enrichments and fractional synthetic rates (0.45 +/- 0.03 day-1, t1/2 = 1.53 +/- 0.09 days) were calculated. Biosynthetic rates of rat serum albumin were congruent with previously published values. In summary, measurement of protein synthesis and precursor pool enrichments by MIDA is technically feasible and practical in vivo using proteolytically derived peptides and ESI-MS analysis., (Copyright 1999 Academic Press.)

In this work, micromachining technologies are employed to develop a miniaturized electrical field-flow fractionation (EFFF) separation system. EFFF systems are used to separate colloidal particles such as cells, liposomes, proteins, or other particulates, and to characterize emulsions and other mixtures according to particle charge density. Macromachining techniques have been used to develop existing EFFF technologies. At the present time, the limiting factor in the development of higher precision EFFF separation systems has been the manufacturing approach. In this paper, the theory behind the operation and resolution of a micron-sized EFFF (mu-EFFF) system is described and the advantages to be gained from application of micromachining technologies are given, thus motivating the need for further miniaturization. A completely fabricated mu-EFFF system is developed, separations are performed, and the mu-EFFF system is compared to the theoretically predicted results as well as the results from current macro EFFF systems.

Thin-layer chromatography (TLC) is one of the simplest and most convenient techniques to separate small molecules. Of a variety of TLC separation modes, only size-exclusion was successfully used to separate proteins. In this paper, adsorption-TLC was used to separate proteins. The net charges were calculated for four model proteins, albumin, transferrin, lactoferrin and lysozyme, under different pH values. The suitable pH values for separation were determined according to the results from such calculations. Then, the adsorption isotherms of the four proteins were measured to deduce the ionic strength for appropriate elution conditions. Optimal conditions, 0.01 M bicine and pH 8.50, and a three-step elution process (1st step 0.01 M NaCl, 2nd 0.025 M NaCl, and 3rd 0.10 M NaCl), were obtained. Finally, the four model proteins were successfully separated under these elution condition.

This study examines the size and compositional heterogeneity of particles in a commercial lipid emulsion (Intralipid) before and after equilibration with penclomedine, a highly lipophilic cytotoxic agent. Emulsions were fractionated by sedimentation field-flow fractionation (sedFFF), and particle sizes of the monodisperse fractions were determined by photon correlation spectroscopy. The triglyceride (TG), phosphatidylcholine (PC), and penclomedine (in drug loaded emulsions) contents in each fraction were determined by HPLC. The aqueous-entrapped volume within Intralipid was determined to be approximately 10% by size-exclusion chromatography using [3H]mannitol. Thirteen sedFFF fractions collected from the drug free emulsions yielded particles ranging in size from 154 to 423 nm. Total channel recoveries were 89% and 95% for TG and PC, respectively. Apparent particle densities varied significantly with size, suggesting heterogeneity in composition as confirmed by PC/TG mass ratios which varied dramatically. Computer fits of the distribution profiles suggested populations of phospholipid vesicles and oil droplets containing excess phospholipid in addition to classical emulsion droplets. Drug loading induced a significant shift of the predominant triglyceride containing population to a larger particle size. The penclomedine distribution profile closely mimicked that of the TG rather than the PC fraction. These studies suggest the need to consider not only size distribution but also compositional distribution in characterizing parenteral emulsions.

The ability to study and regulate cell behavior at a biomaterial interface requires strict control over material surface chemistry. Perhaps the greatest challenge to researchers working in this area is preventing the fouling of a given surface due to uncontrolled protein adsorption. This work describes a method for coupling peptides to hydrophobic materials for the purpose of simultaneously preventing nonspecific protein adsorption and controlling cell adhesion. A hexapeptide containing the ubiquitous RGD cell-adhesion motif was coupled to polystyrene (PS) via a polyethylene oxide (PEO) tether in the form of a modified PEO/PPO/PEO triblock copolymer. Triblocks were adsorbed onto PS at a density of 3.3 +/- (5.14 x 10(-4)) mg/m2 (1.4 x 10(5) +/- 2.12 x 10(1) molecules/microm2), which was determined by isotope 125I labeling. The peptide, GRGDSY, was activated at the N terminus with N-Succinimidyl 3-(2-pyridyldithio) propionate and coupled to immobilized triblocks where the terminal hydroxyls had been converted to sulfhydryl groups. Surface peptide density was measured by amino acid analysis and found to be 1.4 x 10(4) +/- 0.47 x 10(4) molecules/microm2. PS modified with PEO/PPO/PEO copolymers alone was found to be inert to cell adhesion both in the presence of serum proteins and when exposed to activated RGD peptide. In contrast, PS conjugated with RGD via endgroup-activated PEO/PPO/PEO copolymers supported cell adhesion and spreading. The surface coupling scheme reported here should prove valuable for studying cell-ligand interactions under simplified and highly controlled conditions.

Study Objective: The tuberculin skin test is the best diagnostic method to detect tuberculous infection. How accurate is interpretation of the test?, Design: Observational study., Setting: Both general hospital and university hospital., Participants: One hundred seven health-care professionals, including 52 practicing pediatricians, 33 pediatric house officers, 10 pediatric academicians, 11 registered nurses, and 1 pediatric nurse practitioner., Study: A tuberculin skin test (Mantoux) was applied to the arm of a known tuberculin converter. As participants entered/left the room, they were guided to the tuberculin converter. At no time did a participant observe readings other than his/her own., Results: Mantoux tuberculin reaction measuring 15 mm induration was read individually by a group of 52 practicing pediatricians, 33 pediatric house officers, 10 pediatric academicians, 11 registered nurses, and one pediatric nurse practitioner. The median induration recorded by this group of 107 health-care professionals was 10 mm, and 17 (33%) practicing pediatricians read the reaction as <10 mm induration. Using the > or = 15-mm induration indicator to identify a positive reaction, 93% of those in the study (99/107 participants) would have identified our known converter as tuberculin negative., Conclusion: This study confirms a general inaccuracy in interpretation of the tuberculin skin test reaction. It raises two questions. (1) Is there a general tendency toward underreading? (2) Does this general tendency to underread tuberculin skin test reactions raise some question as to the American Academy of Pediatrics, American Thoracic Society, and Centers for Disease Control and Prevention move in raising the amount of induration considered tuberculin positive to 15 mm in low-risk individuals?

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcepsilonRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcepsilonRI cross-linking activates both PLCgamma1 and PLCgamma2. Activation is accompanied by the increased phosphorylation of both PLCgamma isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCgamma isoforms have distinct subcellular localizations. PLCgamma1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCgamma1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCgamma2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcepsilonRI cross-linking. The activation of PLCgamma1, but not of PLCgamma2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCgamma1 with little inhibition of PLCgamma2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCgamma1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCgamma1. Although less abundant than PLCgamma2, activated PLCgamma1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.

We measured 13 metals in the urine of 496 United States residents to establish reference range concentrations using inductively coupled argon plasma mass spectrometry and Zeeman graphite furnace atomic absorption spectrometry. We frequently found 8 of these analytes at detectable concentrations in urine specimens: molybdenum (in 99.8%); lead (98.8%); tin (89%); thallium (77%); antimony (73.5%); manganese (73%); cesium (71%); tungsten (70%); and platinum (69.7%). The 95th percentile concentration for molybdenum was 168 micrograms/L; concentrations ranged up to 688 micrograms/L. Lead concentrations ranged up to 67 micrograms/L, and the 95th upper percentile was 6.4 micrograms/L. Tin had 95th upper percentile of 20.1 micrograms/L. Other analytes measured at detectable concentrations included barium (in 67% of the specimens); beryllium (67%); chromium (54%); thorium (44%); and cobalt (43%). In almost every case, the 95th upper percentiles of these analytes were less than 15 micrograms/L.

Two samples of sixth-grade students were followed over time to examine relations of number of reciprocated friendships, peer acceptance, and group membership to academic achievement. In both samples, group membership was the most consistent predictor of grades over time. In Study 2, prosocial behavior, antisocial behavior, and emotional distress were examined as processes that might explain these significant links between peer relationships and academic achievement. Results of longitudinal analyses support a conclusion that aspects of peer relationships are related to classroom achievement indirectly, by way of significant relations with prosocial behavior. Future research might benefit from more in-depth analyses of the functions of adolescent peer relationships and the processes by which they influence orientations toward social and academic competence at school.

To identify regulators of stem cell lineages, we are focusing on spermatogenesis in Drosophila. In spermatogenesis, each germline stem cell divides asymmetrically, renewing itself and producing a transiently amplifying daughter, which divides four times. By screening for mutants in which daughter cells fail to stop dividing, we find that the TGF-beta signal transducers schnurri and punt are required to limit transient amplification of germ cells. Mosaic analysis demonstrates that punt and schnurri act within somatic cyst cells that surround germ cells, rather than in germ cells. Thus, a cyst-cell-derived signal restricts germ cell proliferation and this signal is initiated by input from a member of the TGF-beta superfamily. Thus, a signal relay regulates progression through the germline stem cell lineage.

Previous studies in our laboratory using a rat model of fetal alcohol exposure (FAE) suggest that FAE-induced behavioral deficits are, in part, linked to neurochemical and electrophysiological deficits in long-term potentiation (LTP) in the entorhinal cortical perforant path projection to the hippocampal formation. Several findings suggest that signal-activated phospholipase C (PLC) and phospholipase A2 (PLA2) are critical to the induction and maintenance of LTP. Thus, alterations in phospholipid metabolism may play a significant role in the LTP deficits observed in FAE offspring. To test this hypothesis, we measured PLC-beta 1 and PLA2 activities in the hippocampus and medial frontal cortex of adult rats prenatally exposed to ethanol. PLC-beta 1 activities were significantly decreased by 20 to 30% in both the hippocampus and medial frontal cortex of FAE rats, compared with ad libitum and pair-fed controls. Total Ca(2+)-dependent PLA2 activity was 25% lower in the medial frontal cortex of FAE rats, but did not significantly differ from controls in the hippocampal formation. Approximately 30% of the measured activity in both the medial frontal cortex and hippocampal formation of ad libitum and pair-fed animals was associated with an 85 kDa cytosolic PLA2 form. Cytosolic PLA2 activities were significantly reduced in both the medial frontal cortex and hippocampal formation of FAE rats, compared with controls. These changes in Ca(2+)-dependent PLA 2 and PLC-beta 1 activities, coupled with reports of FAE-induced deficits in protein kinase C activity, indicate that prenatal exposure to moderate quantities of ethanol causes profound and long-lasting deficits in the cellular signaling mechanisms associated with activity-dependent synaptic plasticity and memory formation.

The development of curricula in nurse education cannot be viewed in isolation from the socio-political context within which it exists. Changes in the prevailing political ideology have impacted on the social life of individuals, and the reflection of this can be traced in the curricula of nursing courses over time. To ensure that courses meet the needs of both students and society, it is important for teachers to recognize the influence of the context within which such development takes place, and to understand the ideological underpinning of that which is being both taught and learnt. It is only with adequate understanding that challenges can be presented and change promoted when necessary.

Two Chinese hamster ovary cell (CHO-K1) mutants selected for defective glutamate transport via system X-AG are also highly permeable to small neutral molecules. Light microscopy demonstrated that exposure of one of these mutants, Ed-A1, to hypo-osmotic medium led to extremely rapid swelling, presumably due to increased water flux. When placed in 20% saline, Ed-A1 cells swelled to three times their original volume within 15 sec, a sixfold larger increase than parental CHO-K1. In spite of this rapid volume increase, mutant and wild-type cells remained viable for 20 min in dilute saline. A regulatory volume decrease in Ed-A1, and the continual swelling of CHO-K1, resulted in the two cells achieving equal size after 5 min in 20% saline. The time course of these volume changes permitted analysis of large numbers of cells by a hydrodynamic technique, steric field flow fractionation (FFF). Steric FFF demonstrated the expected inhibition of osmotic swelling of human erythrocytes by the mercurial, p-chloromercuribenzenesulfonic acid (PCMBS). However, PCMBS increased the apparent swelling rate of Ed-A1 and CHO-K1, suggesting that an aquaporin-like molecule is not responsible for any significant fraction of the water fluxes into either line. PCMBS also strongly inhibited aspartate transport by system X-AG. By taking advantage of their different swelling rates in hypotonic medium, steric FFF can separate mixtures of CHO-K1 and Ed-A1.

A general route has been developed to chemically modify a series of poly(ethylene oxide)-poly(propylene oxide) triblock copolymers with molecular weights from 6500 to 14600. It is initiated by the introduction of p-nitrophenyl groups; such nitrophenyl conjugated copolymers are stable in an organic milieu and in a dry state but are seen to react easily with amino-containing molecules including small peptides. Among them, introduction of 2-pyridyl disulfide groups after coupling with 2-(2-pyridyldithio)ethylamine enables the selective attachment of thiol-containing molecules. The released thiopyridone in such thiol-disulfide reactions can be used to quantify the content of 2-pyridyl disulfide groups. In addition, a new type of modified copolymers was developed for the radioisotope (125I) labeling purpose that consists of a reaction of nitrophenyl conjugated copolymers with hydrazine and a subsequent coupling with N-succinimidyl 3-(4-hydroxyphenyl)propionate (Bolton-Hunter reagent). Adsorption studies of 125I-labeled and 2-pyridyl disulfide conjugated copolymers on polystyrene particles are consistent with previous determinations of surface coverage using other technologies, in turn indicating that this new chemical modification does not alter their surfactant properties on hydrophobic solid phase. The coating of common hydrophobic surfaces with 2-pyridyl disulfide conjugated copolymers has been demonstrated as a general and robust immobilization method to generate a high-sensitivity bioactive surface with low nonspecific binding. The optimal space between immobilized ligands can also be controlled by incubating the solid phase with solutions containing mixtures with different ratios of unmodified and modified copolymers.

The binding of 5'-end biotinylated DNA, ranging in size from 100 to 5000 base pairs, was studied using streptavidin-coated polystyrene latex particles with diameters between 0.944 and 0.090 micron. The experimental binding constants and forward rate constants of this solid-phase reaction were determined to be several orders of magnitude lower than values for the biotin-streptavidin interaction in solution as expected and were shown to depend on the size of both ligand and substrate. An observed inflection in the binding constant of the biotinylated DNA appeared around 1000 base pairs, possibly indicating different surface orientations of the macroligand above and below this critical size. This effect was more pronounced for the smaller latex particles used in this study and highlighted possible differences in the surface arrangement of streptavidin on the differently sized particles. Diffusion limitation to the binding reaction was found to be significant in all cases. In this present work, an exponential relationship was established between the experimental binding constant and the number of base pairs in the biotinylated DNA. This relationship possibly provides a means to predict capacity and binding speed in cases where adsorption, purification, and release of larger DNA chains are required.

Objective: To compare the subclass distribution of anti-48 kDa La(SSB) and anti-52 and 60 kDa Ro(SSA) antibodies in the maternal and neonatal circulation, in pregnancies affected and unaffected by the development of congenital heart block (CHB)., Methods: Sera were obtained from 32 mothers (during 34 pregnancies 23 complicated by CHB and 11 healthy) demonstrated to have anti-Ro(SSA) and/or La(SSB). Maternal and neonatal autoantibodies were evaluated for subclass distribution by ELISA:, Results: All 4 subclasses of anti-Ro(SSA) and La(SSB) antibodies cross the placenta and are detectable in sera obtained from the umbilical cord, IgG1 and IgG3 were the major subclasses represented in the 48 kDa La(SSB) and 52 kDa Ro(SSA) responses. All subclasses, including IgG2 and IgG4, were observed in about one-third of the anti-52 kDa Ro(SSA) and 48 kDa La(SSB) responses. In contrast, anti-60 kDa antibodies were, with rare exception, confined to IgG1. Except for anti-48 kDa La(SSB) IgG3 antibodies, no significant differences were observed between affected and unaffected pregnancies in the ratio of maternal to neonatal levels of any of the antibody subclasses. Overall, there were no significant differences in the subclass profiles between mothers whose children had heart block and those who did not., Conclusion: The IgG subclasses of anti-48 kDa La(SSB) and anti-52 and 60 kDa Ro(SSA) do not account for the susceptibility of one fetus versus another for the development of CHB. Anti-60 kDa Ro(SSA) antibodies are more restricted in subclass distribution than anti-52 kDA Ro(SSA) or 48 kDa La(SSB) responses.

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis.

Problems of living with an altered body image following stoma surgery have long been recognised, but the specific needs of homosexual patients undergoing such surgery have been largely disregarded. This article examines the ways in which nurses can support their homosexual stoma clients.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.

Purpose: To investigate the efficacy and safety of oral ondansetron in the control of cisplatin-induced delayed emesis in patients who do not require rescue antiemetic therapy for acute emesis., Patients and Methods: Five hundred thirty-eight chemotherapy-naive patients who received cisplatin chemotherapy (> or = 70 mg/m2), and who were not rescued for acute emesis, were eligible to be randomized to receive one of the three oral regimens to control delayed emesis. Group I received placebo on days 2 to 6; group II received ondansetron 8 mg twice daily on days 2 and 3 and placebo on days 4 to 6; group III received ondansetron 8 mg twice daily on days 2 to 6. All patients received intravenous ondansetron (0.15 mg/kg every 4 hours for three doses) for the control of acute emesis on day 1. The number of emetic episodes on days 2 and 3 combined (days 2/3, when incidence and severity of delayed emesis were expected to be greatest) was considered the primary measure of efficacy., Results: Patients who received odansetron had significantly fewer emetic episodes on days 2/3, 4, and 5 than those who received placebo (P < or = .002 on each day). Additionally, significantly more patients who received ondansetron had a complete plus major response (C+MR; < or = two two emetic episodes) than those who received placebo on days 2/3 (56% v 37%, P = .001), 4 (94% v 85%, P = .005), and 5 (98% v 88%, P = .006). Patients who received ondansetron had significantly less nausea on day 2/3 when day-1 nausea was used as the baseline score (P = .025). Patients who received ondansetron also had significantly less nausea on day 4 (P = .042) and the results approached significance on day 5 (P = .066)., Conclusion: Oral ondansetron had a significant effect in the control of cisplatin-induced delayed emesis and nausea in patients who had not required rescue antiemetics during the acute emesis period. The control of delayed nausea and vomiting was most notable in the immediate 2 days following cisplatin administration, with the clinical difference narrowing between the two treatment arms on subsequent days.

Little is known about the control of events ending the lengthy prophase of meiosis I and leading to the G2/M-phase transition in mammalian spermatocytes, primarily because the relevant late pachytene, diplotene, and MI cells are present in low numbers in the testis and it is not possible to isolate them in significant numbers. We have utilized short-term cultures of pachytene spermatocytes from the mouse to study events of the G2/M cell-cycle transition induced by the protein phosphatase inhibitor okadaic acid (OA). Treatment of cultured pachytene spermatocytes with OA induced a rapid and premature onset of events leading to the M phase, visualized cytologically by nuclear envelope breakdown and chromosome condensation. After OA treatment, condensed chromosomes were seen as bivalents, not as univalents. Treatment with OA induced disassembly of synaptonemal complexes and resolution of crossovers as cytologically visible chiasmata. Chiasmata counts were similar in treated cells and control cells. Thus, surprisingly, even though the treated cells were in the pachytene substage of meiotic prophase, events of recombination were apparently completed to the point of chiasma formation in the majority of these cells. The sex chromosomes, forming the sex body of the pachytene spermatocyte, lagged behind the autosomal chromosomes in their condensation and progression toward the M phase. Treatment with OA induced an increase in histone H1 kinase activity, generally used as an indicator of metaphase-promoting factor (MPF) activity; furthermore, the OA-induced cell-cycle transition does not require new protein synthesis. These results suggest that OA treatment overrides a cell-cycle checkpoint control that normally keeps pachytene spermatocytes in a lengthy prophase and that this control may be exerted by regulation of protein phosphorylation status.

The binding of 5'-end biotinylated DNA fragments was compared between streptavidin (SA)-coated commercial M-280 magnetic latex particles with a diameter of 2.8 microns and adsorption-coated polystyrene (PS) latex standard particles whose diameter is 0.272 microns. Amino acid analysis showed the protein content of the commercial particles to correspond to 4x monolayer coverage, while the adsorption-coated PS particles displayed monolayer coverage, or 8 pmol/cm2. A fluorescence-based method was developed to quantify the adsorption of FITC-labeled SA, biotin, and biotinylated DNA. The validity of the method was substantiated for the labeled protein by both amino acid analysis and a colorimetric protein assay. While the specific binding of biotin was 0.38 mol per mole of SA on the adsorption-coated 0.272-microns particles and slightly higher (0.6 mol per mole SA) on the 2.8-microns particles, the specific binding of the bulky biotinylated 300-bp DNA was more favorable on the smaller particle (0.12 mol per mole SA for 0.272 microns versus 0.04 mol per mole SA for 2.8 microns).

In this paper, we demonstrate that by employing a combination of sedimentation field-flow fractionation (sedFFF) and other characterization techniques, such as photon correlation spectroscopy (PCS) and freeze-fracture electron microscopy (EM), it is possible to show that commercial fat emulsions of similar overall chemical compositions not only may exhibit different size distributions but may have different densities as well. A closer look at the density difference between droplet and suspension medium, on the one hand, and the droplet size, on the other, demonstrates that fat emulsions may have structures other than the traditional oil droplet surrounded by a monolayer of surfactant. From our determined and simulated density differences, we propose that these emulsion droplets may have a multilayered surfactant arrangement as well as an inclusion of water vesicles in the oil phase of the emulsion. Freeze-fracture EM observations provide evidence to confirm the existence of such complex structures. These findings are supported by recent EM work from other laboratories, as well as through chemical verification of elevated water contents in the oil droplets of these emulsions.

Objective: Since there is suggestive but, to date, indirect evidence that maternal anti-SSA(Ro) and SSB(La) antibodies are pathogenic in congenital heart block (CHB), we explored the hypothesis that binding to fetal tissues would result in selective depletion of autoantibodies from the neonatal circulation., Methods: Maternal and umbilical cord levels of anti-48 kDa SSB(La), anti-52 kDa SSA(Ro) and anti-60 kDa SSA(Ro) antibodies were measured by ELISA and immunoprecipitation at parturition in 15 pregnancies complicated by CHB. A control group consisted of 13 pregnancies in which the mother was known to have antibodies to either SSA(Ro) and/or SSB(La) and the children did not have CHB., Results: The ratios of maternal to cord serum levels of anti-48 SSB(La), anti-52 SSA(Ro) and anti-60 kDa SSA(Ro) antibodies ranged from 0.71 to 2.38 in both affected and unaffected pregnancies. The mean ratio obtained for each of the 3 autoantibodies was not significantly different between the 2 groups. Moreover these ratios did not significantly differ from the mean ratios obtained for total IgG levels in either group., Conclusion: These data demonstrate that maternal antibodies to all components of the SSA(Ro) SSB(La) system are efficiently transported across the placenta and are not selectively depleted in the circulation of neonates with CHB.

Aromatic β-hydroxyoximes undergo unusual fragmentation reactions as protonated or cationized species, as radical cations, or as (M - H)(-) ions, As protonated species, they expel OH ' from the oxime functionality in violation of the even electron rule. Parallel eliminations of alkyl radicals follow OH' loss when the aromatic ring is substituted with an alkyl chain. Alkyl radical losses appear to be characteristic of radical cations that can isomerize to ions in which the alkyl chain bears a radical site and the charged site is the conjugate acid of a basic functionality (e.g., oxime or imine). Evidence for the mechanisms was found in the ion chemistry of oxime and imine radical cations. The imine reference compounds were conveniently generated by fast atom bombardment-induced reduction of oximes, removing the requirement for using conventional chemical synthesis. Protonated imines and the (M - H)(-) ions of oximes fragment extensively via charge-remote processes to eliminate the elements of alkanes. This chemistry is not shared by the protonated oximes.

In its original implementation, electrical field-flow fractionation (EFFF) was carried out in membrane-walled channels with the electrodes placed externally to the flow channel. The poor separation efficiency of this system left the technique largely unattended for about two decades. In the present study, we describe a new and simple approach to EFFF, which demonstrates the technique's ability to carry out rapid, high-resolution separations of colloidal samples in aqueous suspensions. The present channels are bounded by the smooth and rigid graphite electrodes which allow for the application of small voltages, typically less than 2 V, across the thin (178 microns) separation space defined by a Mylar spacer. Although this arrangement generates nominal fields of the order of 100 V/cm, polarization of the electrodes considerably reduces the effective field across the bulk of the channel to less than 1% of the nominal value. Nevertheless, under conditions of low ionic strength the system is shown to retain and separate polystyrene (PS) latex standards with sizes ranging from 60 to 10,000 nm. For small particles of comparable zeta-potential, separating in the "normal" mode of EFFF, the size selectivity Sd, is approximately 0.7. As with other FFF techniques, EFFF displays a transition from "normal" to "steric" behavior; the critical diameter for this transition is highly dependent on ionic strength, with values ranging from approximately 500 nm in deionized water to approximately 1200 nm in 133 microM aqueous NaCl under an applied voltage of 1.37 V and a flow of 1 mL/min.(ABSTRACT TRUNCATED AT 250 WORDS)