Buffy coat volume reduction for optimization of leucapheresis harvests produced by the autoMNC program.

BACKGROUND AND OBJECTIVES: Buffy coat (BC) volume reduction was evaluated in leucapheresis (LA) harvests due to the target monocyte yield and the red blood cell (RBC) content. A packed erythrocyte volume (PEV) of 7.5 ml should not be exceeded to avoid RBC debulking with loss of leucocytes (WBCs) and the monocyte fraction during monocyte counterflow elutriation, a next step of monocyte enrichment prior to cell culture. MATERIALS AND METHODS: Two hundred and fifty-three 5-l leucaphereses (autoMNC program) performed in 102 healthy blood donors (24 female and 78 male donors) were retrospectively analysed. Different categories of BC volumes were compared due to the quality of the LA products measured by blood counts and flow cytometry. RESULTS: Collection of maximum BC volume of 10 ml and more each collection cycle (product volume: 169 ± 21 ml) resulted in 1.58 ± 0·41 × 10e9 CD14(+) monocytes and high volume of packed erythrocyte (18.4 ± 8.8 ml). Low BC volume collection below 6 ml each collection cycle produced only 1.07 ± 0.40 × 10e9 CD14(+) monocytes but reduced PEV significantly by 64% (6.7 ± 4.1 ml). CONCLUSION: By reduction of the BC volume, the PEV in LA products could be reduced, which is a precondition for counterflow elutriation of monocytes. A BC volume between 7 and 8 ml per collection cycle should be adjusted to reduce PEV to 7.5 ml without relevant monocyte loss.

Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors.

The Ras(17N) dominant negative antagonizes endogenous Ras function by forming stable, inactive complexes with Ras guanine nucleotide exchange factors (GEFs; e.g., SOS1). We have used the growth-inhibitory phenotype of Ras(17N) to characterize two aspects of Ras interaction with GEFs. First, we used a nonprenylated version of Ras(17N), designated Ras(17N/186S), which no longer associates with the plasma membrane and lacks the growth-inhibitory phenotype, to address the importance of Ras subcellular location and posttranslational modification for its interaction with GEFs. We observed that addition of an N-terminal myristylation signal to Ras(17N/186S) restored the growth-inhibitory activity of nonprenylated Ras(17N). Thus, membrane association, rather than prenylation, is critical for Ras interaction with Ras GEFs. Second, we used a biological selection approach to identify Ras residues which are critical for Ras(17N) growth inhibition and hence for interaction with Ras GEFs. We identified mutations at residues 75, 76, and 78 that abolished the growth-inhibitory activity of Ras(17N). Since GEF interaction is dispensable for oncogenic but not normal Ras function, our demonstration that single-amino-acid substitutions at these three positions impaired the transforming activity of normal but not oncogenic Ras provides further support for the role of these residues in Ras-GEF interactions. Finally, Ras(WT) proteins with mutations at these residues were no longer activated by mammalian SOS1. Altogether, these results suggest that the Ras intracellular location and Ras residues 75 to 78 are critical for Ras-GEF interaction.

Analysis of the 5' end of the rat plasma membrane Ca(2+)-ATPase isoform 3 gene and identification of extensive trinucleotide repeat sequences in the 5' untranslated region.

We have characterized the 5' end of the rat gene encoding isoform 3 of the plasma membrane Ca(2+)-ATPase using S1 nuclease protection and DNA sequence analysis. The 5'-untranslated region consists of over 900 nucleotides and includes a 217-nucleotide sequence composed of alternating tracts of TCC and ACC trinucleotides. Analysis of genomic sequences 5' to the transcription initiation site revealed potential binding sites for transcription factors that are active in muscle and brain.

Plasma glucose levels are reduced in rats and mice treated with an inhibitor of glucose-6-phosphate translocase.

The activity of glucose-6-phosphatase (G-6-Pase) in isolated rat microsomes was inhibited by a new selective inhibitor of the multi-subunit G-6-Pase system, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3,4-dihydroxy-5-(3-imid azo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (compound A) with a 50% inhibitory concentration (IC50) of approximately 10 nmol/l. Compound A (500 nmol/l) inhibited the uptake of [14C]glucose-6-phosphate (G-6-P) into intact isolated rat microsomes, confirming that this agent blocks G-6-P translocation, as suggested by previous studies using intact and permeabilized microsomes. The inhibition of microsomal G-6-P transport by compound A was associated with inhibition of the rate of glucose output from rat hepatocytes incubated in the presence of 25 nmol/l glucagon (IC50 approximately 320 nmol/l.) Compound A (1 micromol/l) also inhibited the basal rate of glucose production by rat hepatocytes by 47%. Intraperitoneal administration of compound A to fasted mice lowered circulating plasma glucose concentrations dose-dependently at doses as low as 1 mg/kg. This effect was comparatively short-lived; glucose lowering was maximal at 30 min after dosing with 100 mg/kg compound A (-71%) and declined thereafter, being reversed within 3 h. A similar time course of glycemic response was observed in fasted rats; glucose lowering was maximal 30 min after dosing with 100 mg/kg compound A (-36%) and declined until the effect was fully reversed by 3 h postdose. In rats subjected to compound A treatment, liver glycogen content was increased. G-6-P and lactate levels were maximally elevated 30 min after dosing and declined thereafter. Cumulatively, these results suggest that the mechanism of glucose lowering by compound A was via inhibition of G-6-Pase activity, mediated through inhibition of the T1 subunit of the microsomal G-6-Pase enzyme system. Drug levels measured over the same time course as that used to assess in vivo efficacy peaked within 30 min of administration, then declined, which is consistent with the transient changes in plasma glucose and liver metabolites.

Biomolecular applications of heteronuclear multidimensional NMR.

Recent advances in stable-isotope enrichment and heteronuclear multidimensional NMR techniques have transformed NMR into a more powerful and versatile method for the structural and dynamic characterization of biomolecules. Current efforts still focus on improving the methodology to increase the number of systems amenable to NMR analysis, to generate better structures and to investigate biomolecular motions in solution.

Cataract surgery combined with implantation of an artificial iris.

We describe 6 patients who presented with cataract or aphakia and absent or nonfunctional irides. The etiologies included congenital aniridia, traumatic iris loss, and chronic mydriasis secondary to recurrent herpetic uveitis. In 5 eyes, a prosthetic iris was successfully implanted in combination with small incision cataract surgery. In 2 eyes, a single-piece iris diaphragm and optical lens was implanted. Artificial irides offer a safe alternative for patients who previously had no viable options for iris reconstruction.

Prosthetic iris implantation for congenital, traumatic, or functional iris deficiencies.

PURPOSE: To determine the efficacy and safety of surgical implantation of prosthetic iris devices in patients with anatomic or functional iris deficiencies. SETTING: Cincinnati Eye Institute, Cincinnati, Ohio, USA. METHODS: Twenty-five patients were enrolled in an interventional prospective noncomparative case series. Twenty-eight eyes had prosthetic iris diaphragm implantation for traumatic iris defects, congenital aniridia or iris coloboma, herpetic iris atrophy, surgical iris loss, or ocular albinism. Prosthetic iris implantation was performed with phacoemulsification and intraocular lens (IOL) implantation in 20 eyes, secondary IOL implantation in 6 eyes, and IOL exchange in 1 eye. A single pseudophakic eye with disabling glare secondary to traumatic aniridia had secondary prosthetic iris implantation alone. The surgical ease of insertion, intraoperative and postoperative complications, postoperative anatomic results, visual acuity, and subjective glare reduction were evaluated. RESULTS: Patients were followed postoperatively for a mean of 10.2 months (range 1.4 to 25.7 months). All eyes achieved the desired anatomic result. Visual acuity was improved in 22 of 28 eyes (79%), unchanged in 5 eyes, and worsened by a single line in 1 eye. Patients were surveyed postoperatively to determine the change in glare disability. The severity of glare disability was subjectively improved in 23 of 24 patients (96%) who responded to the survey. Intraoperative complications included 3 fractured implants as well as an incomplete or torn capsulorhexis in 3 eyes. Postoperative complications included transient hypotony in 2 eyes, mild persistent inflammation in 1 eye, and macular edema followed by a retinal detachment in 1 eye with recent severe trauma. CONCLUSIONS: Implantation of prosthetic iris devices improved postoperative outcomes by reducing glare disability and, in selected cases, by correcting aphakia. Although operating on traumatized, congenitally aniridic, or uveitic eyes presents special challenges, implantation of prosthetic iris devices appears to be a safe and effective method for reducing the ubiquitous glare in patients with iris deficiency.

Monoclonal antibodies directed against tick-borne encephalitis virus with neutralizing activity in vivo.

Monoclonal antibodies (MoAbs) were raised against the tick-borne encephalitis (TBE) virus, strain K23. The reactivities of 14 selected MoAbs were characterized by ELISA, Western blot analysis, haemagglutination inhibition, immunoprecipitation, in vivo protection and in vitro neutralization tests. All MoAbs reacted only with the glycoprotein E. The binding epitope of one MoAb could be delimited by a synthetic peptide to amino acids 306-339 representing one immunodominant loop structure of the glycoprotein E. The MoAbs exhibited individual reactivities against 13 different TBE virus isolates in ELISA and immunoblot test ranging from type-specific reactions to a broad reactivity with all isolates. Four MoAbs also showed a cross-reaction with other flaviviruses like West Nile virus and/or Yellow fever virus in immunoblot analysis. By competition ELISA the MoAbs could be divided into five different reaction patterns. Four MoAbs showed neutralizing activity with titers in the range 1:140 to 1:5,000 in an in vitro assay. These neutralizing activities could be confirmed by an in vivo mouse challenge model. The MoAbs are useful for diagnostic purposes and for differentiation of TBE virus strains and other flaviviruses.

Treatment of acute migraine with sumatriptan--response in 40 consecutive patients.

Forty consecutive headache patients self-administered sumatriptan for migraine, diagnosed by the criteria of the International Headache Society (IHS). Eighty percent reported excellent response. Thirty-three percent had recurrence of headache within four to twelve hours, while 67% had no recurrence. Fifty-five percent of the patients reported mild side effects, but only 8% stopped therapy because of adverse reactions. No serious cardiovascular events occurred. Recommendations for safe use of sumatriptan are suggested.

The effects of windrow composting on the viability of Parascaris equorum eggs.

Parascaris equorum generally infects horses less than 18 months old and its pathological effects can be severe. Infection occurs when larvated eggs, present in pastures, paddocks, stalls, and on feeding and watering equipment are ingested. The purpose of this study was to examine the effects of windrow composting on the viability of P. equorum eggs at a cooperating central Kentucky horse farm. Three grams of feces containing 2216 P. equorum eggs per gram were sealed in filter bag sentinel chambers. Chambers were exposed to 1 of 3 treatments: constant exposure or intermittent exposure to the interior of the windrow; controls were stored at 4°C. At day 0, all chambers in the experimental treatments were placed in the center of 10 locations of the windrow. On subsequent days when the windrow was turned, chambers in the constant exposure treatment were returned to the interior of the windrow and chambers in the intermittent exposure treatment were alternated between resting on top of, or inside, the windrow. Chambers from each treatment and control chambers were removed at days 2, 4, 6, 8, 10, 12, 14, and 18; and incubated for 21 days at room temperature (24°C). After incubation, eggs were recovered from the chambers using double centrifugation flotation. Eggs were evaluated microscopically, staged according to development and classified as viable or nonviable based on whether embryonation to the larval stage had occurred. Results were reported as the mean percent viable eggs for each treatment and time point. A mixed linear model with repeated measures was used to evaluate the influence of experimental day and treatment on the percent viability of P. equorum eggs. Chambers treated with constant exposure contained 10.73% (SD=0.29) viable eggs on day 2 and declined to an average of 0.00% by day 8. Chambers exposed to the intermittent treatment contained 16.08% (SD=0.26) viable eggs on day 2 and decreased to 0.00% by day 6. Control chambers for days 2, 4, 6, 8, 10, 12, 14, and 18 all had viabilities above 79.00%. A significant fixed effect of experimental day (p<0.0001) and compost treatment (p<0.0001) was observed. There was no significant interaction between experimental day and compost treatment (p>0.7459). The results of this study demonstrate that windrow composting was effective at rendering P. equorum eggs nonviable when it was tested under the conditions at a working horse farm.

[The effect of indomethacin and iloprost on the cardiodepressive actions of various anesthetics on isolated atrial preparations from guinea pigs]. / Einfluss von Indometacin und Iloprost auf die kardiodepressiven Wirkungen verschiedener Narkotika am Vorhof-Aurikel-Präparat des Meerschweinchens.

Effects of Indomethacin and Iloprost on the Cardiodepressant Properties of Various Narcotics on Isolated Auricle Preparations from Guinea Pigs. In isolated auricle preparations from guinea pigs several narcotics (hexobarbital, pentobarbital, ketamine, etomidate, urethane) dose-dependently showed cardiodepressive effects. Premedication of the preparations with indomethacin significantly reduced the narcotics-induced changes of the cardiac action. Iloprost, a stable prostacyclin analogon, had the same efficacy. The influence of both substances could be due to an unspecific membrane effect or a positive inotropic action.

Structure of the rat plasma membrane Ca(2+)-ATPase isoform 3 gene and characterization of alternative splicing and transcription products. Skeletal muscle-specific splicing results in a plasma membrane Ca(2+)-ATPase with a novel calmodulin-binding domain.

We have isolated the rat gene encoding isoform 3 of the plasma membrane Ca(2+)-ATPase (PMCA3) and have determined its exon/intron organization. The PMCA3 gene contains 24 exons and spans approximately 70 kilobases. In addition, we have analyzed the splicing and polyadenylation patterns leading to the production of an alternative 4.5-kilobase (PMCA3) skeletal muscle mRNA that differs from the previously characterized 7.5-kilobase brain mRNA (Greeb, J., and Shull, G. E. (1989) J. Biol. Chem. 264, 18569-18576). cDNA cloning, Northern blot hybridization, and polymerase chain reaction analyses of the 4.5-kilobase mRNA demonstrate (i) the inclusion of a novel 68-nucleotide exon (exon 22) that is specific for skeletal muscle and significantly alters the calmodulin-binding domain and (ii) the utilization of an alternative polyadenylation site following exon 23 which eliminates the last coding exon (exon 24) and 3'-untranslated sequence of the 7.5-kilobase mRNA. We have also identified a 42-nucleotide exon (exon 8) that is included in the skeletal muscle PMCA3 mRNAs, but may be either included or excluded in the brain mRNAs. Exon 8 is inserted immediately before the sequence encoding a putative phospholipid binding domain and thus may alter regulatory interactions of the enzyme with acidic phospholipids.

Structural and dynamic differences between normal and transforming N-ras gene products: a 31P and isotope-edited 1H NMR study.

[15N]Glycine was biosynthetically incorporated into normal cellular N-ras p21 and a position 12 transforming mutant, in order to produce p21 proteins containing several site-specific NMR probes at or near activating positions in the guanine nucleotide binding domain. We have previously assigned all five glycine resonances located in loops directly involved in binding of guanosine diphosphate in the wild-type p21 protein [Campbell-Burk, S., Papastavros, M. Z., McCormick, F., & Redfield, A. G. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 817-820]. In this report, the corresponding glycine resonances in the p21 mutant have been assigned, and spectral differences between normal and mutant p21-guanosine diphosphate (p21.GDP) complexes have been investigated. Our combined 1H[15N] and 31P NMR results show that substitution of aspartate for glycine-12 produces perturbations in the phosphoryl binding domain, near the point of the mutation. Although many of the remaining glycines were unaffected, spectral differences were also observed outside the GDP binding domain. Two of the five active-site glycines in wild-type p21.GDP have very slow amide proton exchange rates with water (kappa less than 2.8 x 10(-5) s-1). The active-site glycines are located in solvent-exposed loops, so their apparent solvent inaccessibility may result from strong hydrogen bond formation between glycine amide protons and bound guanine diphosphate and/or other nearby groups in p21.

Alternative splicing of exons encoding the calmodulin-binding domains and C termini of plasma membrane Ca(2+)-ATPase isoforms 1, 2, 3, and 4.

Rat plasma membrane Ca(2+)-ATPase (PMCA) mRNAs were examined by S1 nuclease protection (isoform 1) and polymerase chain reaction (isoforms 1, 2, 3, and 4) and the corresponding genes were analyzed to determine the tissue-specific splicing patterns involving exons encoding the calmodulin-binding domains and C termini. Splicing of PMCA1 involves a single 154-nucleotide exon that can be either included or excluded; when the exon is included four different splice donor sites, at positions 87, 114, 152, and 154, can be utilized. PMCA2 mRNAs are generated either by the inclusion of a 172-nucleotide exon, by the inclusion of both the 172-nucleotide exon and a 55-nucleotide exon, or by the exclusion of both exons. Four PMCA3 mRNAs arise by alternative splicing of a 154-nucleotide exon, in patterns that are analogous to those of PMCA1, and additional mRNAs are generated by the inclusion of a 68-nucleotide exon immediately before the 154-nucleotide exon. The simplest splicing pattern occurs in PMCA4, where a single 175-nucleotide exon is either included or excluded. The alternative mRNAs for each of the four genes are expressed in a tissue-specific manner and encode enzyme variants with different combinations of calmodulin-binding domains and C termini.

Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.

A sample of Escherichia coli-expressed human N-RAS-encoded p21, a 21-kDa protein, was selectively labeled with 15N at each of the 14 glycine amide positions. Two-dimensional proton-observe 15N correlation spectra showed one peak for each glycine residue. Five glycine resonances were identified with residues near the nucleotide binding site and provide useful reporters of several oncogene-activating positions. Three of these resonances were assigned to residues 10, 15, and 115 from the spectrum of a sample that was also labeled with [13C]valine. These resonances showed extra splitting or broadening due to the 13C label, which could be eliminated by 13C decoupling. Two other peaks were unambiguously identified as Gly-12 and Gly-13 using a one-dimensional edited nuclear Overhauser experiment and by spectral comparison with an Asp-12 mutant. These assignments have provided several site-specific probes of critical domains in p21.