Virus Size Analysis by Gas-Phase Mobility Measurements: Resolution Limits.

Electrospraying (ES) dissolved viral particles, followed by charge reduction and size analysis with a differential mobility analyzer (DMA), offers a flexible size-analysis tool for small particles in solution. The technique relies on pioneering work by Kaufman and colleagues, commercialized by TSI, and often referred to as GEMMA. However, viral studies with TSI's GEMMA have suffered from limited resolving power, possibly because of imperfections in either the instrument (DMA or charge reduction) or the sample solution preparation. Here, we explore the limits of the resolution achievable by GEMMA, taking advantage of (i) cleaner charge reduction methods and (ii) DMAs of higher resolving power. Analysis of the literature provides indications that mobility peak widths (fwhm) of 2% or less may be achieved by combining careful sample preparation with improved instrumentation. Working with purified PP7 bacteriophage particles small enough to be classifiable by existing high-resolution DMAs, we confirm that fairly narrow viral mobility peaks may be obtained (relative full width at half-maximum fwhm <5%). Comparison of spectra of a given apian virus sample obtained with TSI's GEMMA and our improved instrumentation confirms that one critical limitation is the DMA. This is further verified by narrow peaks from murine parvovirus, norovirus, and encephalomyelitis virus samples, obtained in our improved GEMMA with little sample preparation, directly from infected cell cultures. Classification of purified large (60 nm) coliphage PR772 particles leads to broad peaks, due to both viral degradation and limited intrinsic resolution of the DMAs used to cover the range of such large particles. We conclude that improved DMAs suitable for high-resolution analysis of particles larger than 30 nm need to be developed to determine the intrinsic mobility width of viral particles.

[Effects of soccer-specific strains on the locomotor system]. / Auswirkungen fussballspezifischer Belastungen auf den Bewegungsapparat.

PROBLEMS: Soccer as a Stop-and-Go-sport goes along with a high level of physical strain on the locomotor system. Compared to similar kinds of sports, soccer is characterized by a high prevalence of overloads/injuries in the pelvic region. Since soccer frequently involves one-sided shot-training, modifications in the pelvic statics are possible. METHODS: In a pilot study including 15 football-players-FP (age 26.9 +/- 3.1 yrs; 4.4 +/- 0.4 training units/week+ 1 leaque game) the pelvic statics was measured using the 3-d-recording system CMS70 (Zebris, Germany) directly before and after a defined shot training. The positions of the right and the left posterior superior iliac spines - PSIS were compared. Additionally, the stiffness of selective muscles was analyzed. RESULTS: Before intervention the right PSIS was heightened in 8 FP. In 4 FP the pelvic statics was balanced and in 3 FP the right PSIS was abased. After the shot training, the pelvic statics was balanced in 4 FP. In contrast, 10 FP showed a higher right PSIS and 1 FP had a lower right PSIS. However, modifications of the pelvic statics were detected in all directions. DISCUSSION: Our study demonstrates modification of the pelvic statics by asymmetric soccer-specific strains, but the reactions were individually different. It is possible, that changes in the pelvic statics may lead to changed function or overstrain of advertising muscles.

Interactions of plasma osmolality with arterial and central venous pressures in control of sympathetic activity and heart rate in humans.

Plasma osmolality alters control of sympathetic activity and heart rate in animal models; however, it is unknown whether physiological increases in plasma osmolality have such influences in humans and what effect concurrent changes in central venous and/or arterial pressures may have. We tested whether physiological increases in plasma osmolality (similar to those during exercise dehydration) alter control of muscle sympathetic nerve activity (MSNA) and heart rate (HR) in humans. We studied 17 healthy young adults (7 women, 10 men) at baseline and during arterial pressure (AP) transients induced by sequential injections of nitroprusside and phenylephrine, under three conditions: control (C), after 1 ml/kg intravenous hypertonic saline (HT1), and after 2 ml/kg hypertonic saline (HT2). We continuously measured HR, AP, central venous pressure (CVP; peripherally inserted central catheter) and MSNA (peroneal microneurography) in all conditions. Plasma osmolality increased from 287 +/- 1 mosmol/kg in C to 290 +/- 1 mosmol/kg in HT1 (P < 0.05) but did not increase further in HT2 (291 +/- 1 mosmol/kg; P > 0.05 vs. C). Mean AP and CVP were similar between C and HT1, but both increased slightly in HT2. HR increased slightly but significantly during both HT1 and HT2 vs. C (P < 0.05). Sensitivity of baroreflex control of MSNA was significantly increased vs. C in HT1 [-7.59 +/- 0.97 (HT1) vs. -5.85 +/- 0.63 (C) arbitrary units (au).beat(-1).mmHg(-1); P < 0.01] but was not different in HT2 (-6.55 +/- 0.94 au.beat(-1).mmHg(-1)). We conclude that physiological changes in plasma osmolality significantly alter control of MSNA and HR in humans, and that this influence can be modified by CVP and AP.

Investigation of the mechanism of alkane reductive elimination and skeletal isomerization in Tp'Rh(CNneopentyl)(R)H complexes: the role of alkane complexes.

Experiments are described that provide indirect evidence for the involvement of alkane sigma-complexes in oxidative addition/reductive elimination reactions of Tp'Rh(L)(R)H complexes (Tp' = tris-3,5-dimethylpyrazolylborate, L = CNCH(2)CMe(3)). Reductive elimination rates in benzene-d(6) were determined for loss of alkane from Tp'Rh(L)(R)H, where R = methyl, ethyl, propyl, butyl, pentyl, and hexyl, to generate RH and Tp'Rh(L)(C(6)D(5))D. The isopropyl hydride complex Tp'Rh(L)(CHMe(2))H was found to rearrange to the n-propyl hydride complex Tp'Rh(L)(CH(2)CH(2)CH(3))H in an intramolecular reaction. The sec-butyl complex behaves similarly. These same reactions were studied by preparing the corresponding metal deuteride complexes, Tp'Rh(L)(R)D, and the scrambling of the deuterium label into the alpha- and omega-positions of the alkyl group monitored by (2)H NMR spectroscopy. Inverse isotope effects observed in reductive elimination are shown to be the result of an inverse equilibrium isotope effect between the alkyl hydride(deuteride) complex and the sigma-alkane complex. A kinetic model has been proposed using alkane complexes as intermediates and the selectivities available to these alkane complexes have been determined by kinetic modeling of the deuterium scrambling reactions.

Foveated, wide field-of-view imaging system using a liquid crystal spatial light modulator.

The field-of-view (FOV) of a simple imaging system can be dramatically improved using a liquid crystal spatial light modulator (SLM). A SLM can be used to correct the off-axis aberrations that often limit the useful FOV of an imaging system giving near diffraction-limited performance at much larger field angles than would otherwise be possible. Foveated imaging refers to the variation in spatial resolution across the image caused by using the SLM in this application, and it is useful in reducing bandwidth requirements for data transmission.

Early HIV infection in vivo: branching-process model for studying timing of immune responses and drug therapy.

We propose a stochastic, branching-process model of early events in vivo in human or simian immunodeficiency virus (HIV or SIV) infection and study the influence that the time of appearance of virus-specific antibodies or cytotoxic cells, or of administration of antiretroviral drugs, has on the probability of progression to a chronic infection. In some biological scenarios, our model predicts that a few days' delay in response or intervention would make little difference, while in others it would be highly deleterious. We show that prophylactic efficacy does not require perfect efficiency at neutralizing infectious virus. Data from a trial of PMPA, a potent antiretroviral drug, as post-exposure therapy for SIV infection in macaques, reported by C.-C. Tsai, P. Emau, K.E. Follis, T.W. Beck, R. E. Beneveniste, N. Bischofberger, J.D. Lifson, W.R. Morton (J. Virol. 72 (1998) 4265), provides a test of the model. We show that their observations are consistent with a branching-process without invoking supplementary viral- or host-variability. Finally, most animal trials of antiviral drugs or vaccines use very high viral inoculums; our model demonstrates that in such experiments we risk greatly underestimating the efficacy of these agents.

Biosynthesis and secretion of the mannose 6-phosphate receptor and its ligands in polarized Caco-2 cells.

We have analyzed the transport of newly synthesized mannose 6-phosphate (Man-6-P)-bearing proteins (i.e., lysosomal enzymes) in the polarized human colon adenocarcinoma cell line, Caco-2, by subjecting filter-grown cells to a pulse-chase labeling protocol using [(35)S]methionine, and the resulting cell lysate, apical medium, and basolateral medium were immunoprecipitated with insulin-like growth factor II/Man-6-P receptor (IGF-II/MPR)-specific antisera. The results showed that the majority of secreted lysosomal enzymes accumulated in the apical medium at >2 h of chase and that this polarized distribution was facilitated by the IGF-II/MPR selectively endocytosing lysosomal enzymes from the basolateral surface. Treatment with various agents known to affect vesicular transport events demonstrated that incubations at 16 degrees C or incubations with brefeldin A inhibited the secretion of lysosomal enzymes from both the apical and basolateral surface, whereas treatment with nocodazole selectively blocked apical secretion. In contrast, incubation with NH4Cl or nocodazole had a stimulatory effect on basolateral secretion. Taken together, these results demonstrate that the sorting of Man-6-P-containing proteins into the apical and basolateral secretory pathways is regulated by distinct components of the intracellular trafficking machinery.

On T-cell dynamics and the hyperactivation theory of AIDS pathogenesis.

In an earlier article the author proposed a model of T cell dynamics in which rising activation rates produced falling T-cell counts. Here I show that, in a model of this type, apoptosis and proliferation must nearly balance. Hence, small perturbations of these processes could have large consequences, a fact that may be relevant to AIDS pathogenesis. In addition, I show the model can reproduce all decay rates found by McLean and Michie for cells with radiation-induced chromosome damage, provided that stable damage can proliferate. Memory-cell 'amnesia' is not required. T-cell lifetimes come out much shorter than previously estimated.

The disappearing CD4(+)T cells in HIV infection: a case of over-stimulation?

Infection with the human immunodeficiency virus (HIV) causes a gradual decline in essential immune-system cells called CD4(+)"helper" T cells. These cells are also principal viral targets, but, paradoxically, direct cell-killing does not explain their disappearance. HIV also induces a chronic and increasing state of immune activation. In a mathematical model of normal T-cell kinetics incorporating a cytokine growth factor, increased activation alone explains these T-cell losses, a switch from "naïve" to "memory" phenotype, and certain other features of HIV disease.

Survival of home parenteral nutrition-treated patients: 20 years of experience at the Mayo Clinic.

OBJECTIVE: To present the largest single institutional review of demographics, associated primary diseases, and survival of patients receiving home parenteral nutrition (HPN). MATERIAL AND METHODS: We conducted a retrospective review of medical records of all Mayo Clinic patients treated with HPN between 1975 and 1995. The probability of survival was calculated by using Kaplan-Meier analysis. RESULTS: In the 225 study patients requiring HPN (median age, 51 years), the main underlying primary diseases were as follows: inflammatory bowel disease (IBD) (N = 50), nonterminal active cancer (N = 39), ischemic bowel (N = 35), radiation enteritis (N = 32), motility disorder (chronic pseudo-obstruction) (N = 26), and adhesive intestinal obstruction (N = 18). Other conditions included intestinal and pancreatic fistula, refractory sprue, dumping syndrome, and protein-losing enteropathy. The overall probability of 5-year survival during HPN was 60%. The probability of survival at 5 years based on the primary disease was 92% for IBD, 60% for ischemic bowel, 54% for radiation enteritis, 48% for motility disorder, and 38% for cancer. The probability of 5-year survival stratified by age at initiation of HPN was as follows: younger than 40 years, 80%; 40 through 60 years, 62%; and older than 60 years, 30%. Most deaths during therapy with HPN were attributable to the primary disease. Among the 20 patients who died of an HPN-related cause, 11 deaths were from catheter sepsis, 4 from liver failure, 2 from venous thrombosis, and 2 from metabolic abnormalities. CONCLUSION: Survival of HPN-treated patients is best predicted on the basis of the primary disease and the age at initiation of HPN. Patients with IBD and age younger than 40 years have a better 5-year survival in comparison with other groups. Most deaths during treatment with HPN are a result of the primary disease; HPN-related deaths are uncommon.

Deformed-helix ferroelectric liquid-crystal spatial light modulator that demonstrates high diffraction efficiency and 370-line pairs/mm resolution.

New liquid-crystal media and photoconductor materials are being utilized in spatial light modulators to increase their resolution, diffraction efficiency, speed, and sensitivity. A prototypical device developed for real-time holography applications has shown an 8% diffraction efficiency from a holographic grating with a spatial frequency of 370 line pairs/mm (lp/mm). At 18 lp/mm the device has demonstrated a 31% diffraction efficiency with a 600-micros hologram write time using 400-nJ/cm(2) write beams.

Spontaneous emission lifetime of carriers in a semiconductor microcavity measured by photoluminescence without distortion by reabsorption.

A theory for carrier decay rates and a technique for measuring them are reported. Modification of the spontaneous emission rate of carriers by a semiconductor microcavity is investigated with 100-nm-thick bulk GaAs. Reabsorption makes the cavity-mode photoluminescence (PL) decay much faster than the square of the carrier density. Here reabsorption distortion is avoided by collecting PL that escapes the microcavity directly without multiple reflections: a ZnSe prism glued to the top mirror allows PL to escape at angles beyond the cutoff angle for total internal reflection without the prism. At these steep angles, the stop band of the top mirror has shifted to higher energy, so that it does not impede PL emission. Removal of most of the bottom mirror decreases the true carrier decay rate by only 25%, showing that the large enhancements deduced from cavity-mode PL are incorrect. Fully quantum mechanical computation including guided modes corroborates this conclusion. The prism technique could be used to study carrier dynamics and competition between guided and cavity modes in microcavities below and near threshold.

Expression of insulin-like growth factor (IGF)-I receptors, IGF-II/cation-independent mannose 6-phosphate receptors (CI-MPRs), and cation-dependent MPRs in polarized human intestinal Caco-2 cells.

We have analyzed the surface distribution and functional expression of the insulin-like growth factor I (IGF-I) receptor and the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CI-MPR) in the polarized human colon adenocarcinoma cell line, Caco 2. Domain-selective biotinylation of the apical and basolateral surfaces of Caco-2 cells grown on filter supports revealed a 3-4-fold enrichment of these receptors on basolateral membranes. In addition, the biotinylation studies revealed the presence of the cation-dependent MPR on both membrane surfaces, with a 3.4-fold enrichment on basolateral membranes. Binding of 125I-IGF-I at 4 degrees C confirmed similar higher levels of expression of the IGF-I receptor at the basolateral surface than at the apical surface. Cell surface-specific binding of the iodinated lysosomal enzyme beta-glucuronidase was detected at 4 degrees C on both plasma membrane domains. However, significant uptake of beta-glucuronidase at 37 degrees C was observed only from the basolateral surface. These results indicate that the MPRs and the IGF-I receptor are expressed in a polarized fashion in Caco-2 cells and that the IGF-II/CI-MPR present on apical membranes, unlike the IGF-II/CI-MPR expressed on the basolateral surface, is not functional in endocytosing lysosomal enzymes.

Expression of IGF-II and IGF binding proteins in differentiating human intestinal Caco-2 cells.

The mitogenic and metabolic effects of insulin-like growth factor-II (IGF-II) can be modulated by six distinct IGF binding proteins (IGFBPs). As a first step toward understanding the role of IGFs and their binding proteins in intestinal epithelial cell differentiation, the expression of IGF-II and IGFBPs was characterized in the human colon adenocarcinoma Caco-2 cell line. Northern blot analysis revealed two IGF-II transcripts of 5.4 and 4.5 kb, and ribonuclease protection assays indicated that IGF-II mRNA levels are regulated during Caco-2 differentiation. A specific radioimmunoassay detected IGF-II in serum-free conditioned medium, the level of which was three- to fivefold higher in proliferating cells than in differentiated cells. Immunoprecipitation and ligand blot analyses of conditioned medium demonstrated that IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6 are synthesized by Caco-2 cells, with IGFBP-2 and IGFBP-4 being the major IGFBPs secreted, and that the levels of IGFBP-2 and IGFBP-6 decreased as differentiation proceeded. These results indicate that the expression of IGF-II, IGFBP-2, and IGFBP-6 is regulated in a differentiation-dependent manner in Caco-2 cells.

Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells.

The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes alpha-D-mannosidase, beta-hexosaminidase, beta-glucuronidase, and beta-galactosidase increased approximately 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors (MPRs), the insulin-like growth factor II/cation-independent MPR (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not change significantly during Caco-2 differentiation. In addition, quantitative Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout differentiation, the level of expression of the MPRs was sufficient to target the increased levels of lysosomal enzymes to the lysosome. Unlike the expression of lysosomal enzymes, Western blot analysis demonstrated an approximately 40% and approximately 30% decrease, respectively, in the steady-state levels of free and conjugated ubiquitin during Caco-2 differentiation. Taken together, these results show that the ubiquitin-dependent proteolytic pathway is regulated differently than the lysosomal degradative pathway during Caco-2 differentiation.

The bovine mannose 6-phosphate/insulin-like growth factor II receptor. Localization of the insulin-like growth factor II binding site to domains 5-11.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds two distinct ligands, mannose 6-phosphate (Man-6-P) and insulin-like growth factor II (IGF-II). The extracytoplasmic region of the receptor is composed of 15 homologous repeating domains and domains 1-3 and 7-9 have been shown to contain the two Man-6-P binding sites. To determine the location of the single IGF-II binding site, truncated forms of the M6P/IGF-II receptor were expressed transiently in COS-1 cells and assayed for their ability to bind iodinated human recombinant IGF-II. The binding of [125I]IGF-II to the receptors in the presence or absence of excess unlabeled IGF-II, IGF-I, or insulin was determined by incubation with homobifunctional cross-linking agents followed by SDS-polyacrylamide gel electrophoresis. These binding studies demonstrated that a construct encoding domains 5-11 bound 0.9 mol of IGF-II/mol of receptor, whereas a construct encoding domains 5-10 exhibited no detectable binding to IGF-II. These results indicate that the IGF-II binding site of the M6P/IGF-II receptor is contained within domains 5-11 and that residues in domain 11 play an important role in IGF-II binding.

Optimization of surface micromorphology for enhanced osteoblast responses in vitro.

In vitro cellular responses of osteoblast-like cells were studied on titanium surfaces with different surface morphologies. Surface profilometry was used to determine whether rough or smooth surfaces with regular or irregular morphologies can be produced by conventional fabrication techniques. Significantly higher levels of cellular attachment were found using rough, sandblasted surfaces with irregular morphologies. These results correlate with recent in vivo findings and suggest that implants should be prepared with roughened surfaces at bony contact areas.

Immunohistochemical localization of carcinogen-metabolizing enzymes within the rat and hamster exocrine pancreas.

The P-450 cytochromes, reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase, epoxide hydrolase, and glutathione S-transferases all play important roles in the bioactivation and detoxication of various classes of chemical mutagens and carcinogens. The present investigation was undertaken to determine if and where these enzymes are located within the exocrine pancreas, a tissue that is a target for chemically induced neoplasia. In this study, reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase, two isozymes of cytochrome P-450 (cytochromes P-450 PB-B and BNF-B), epoxide hydrolase, and glutathione S-transferases B, C/A, and E were each localized at the light microscopic level within exocrine pancreases of untreated rats and hamsters utilizing the unlabeled antibody peroxidase-antiperoxidase staining technique. Immunohistochemical staining for each of these enzymes was apparent within acinar cells in pancreases of Holtzman, Sprague-Dawley, Wistar, and Fischer 344 rats. Staining for the reductase, the epoxide hydrolase, and the glutathione S-transferases was also observed within the epithelia of both interlobular and intralobular ducts in the exocrine pancreases of these rat strains, whereas staining for cytochromes P-450 PB-B and BNF-B was not readily detectable within epithelial cells of the rat pancreatic duct system. In the exocrine pancreas of the Syrian golden hamster, immunohistochemical staining for reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase, epoxide hydrolase, and glutathione S-transferases B and C/A was similar to that observed within the rat exocrine pancreas. In contrast, acinar and duct cells in the hamster pancreas both appeared to be stained for cytochrome P-450 PB-B, whereas staining for cytochrome P-450 BNF-B could not be readily detected within either acinar or duct cells, and staining for glutathione S-transferases E did not appear to be present within duct cells in the hamster pancreas. The results of this investigation therefore suggest that highly reactive and toxic electrophilic metabolites of procarcinogens may be generated to the greatest extent within acinar cells in the rat pancreas, whereas these metabolites may be produced within both acinar and duct cells in the hamster pancreas. Regardless of where they are formed, reactive metabolites may be enzymatically detoxicated within both acinar and duct cells in the rat and hamster exocrine pancreas.

Affinity chromatography in nonionic detergent solutions.

Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberatd from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfuly translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.