Signal reversal in Kelvin-probe force microscopy.

Kelvin-probe force microscopy is a measurement mode of atomic force microscopy, which is used to quantitatively map the electrical surface potential of a sample. Inadequate hardware and electronic design can lead to signal cross talk and, in consequence, false results. Here, we show that certain cross talk artifacts not only do manifest themselves in additional noise, reduced resolution, or an offset of the measured surface potential but can also lead to an inverted signal scale and, crucially, cannot be diagnosed with a known reference signal. We show experimental data on an electrically homogeneous sample, describe a method to detect the artifact, and propose simple remedies, which should be well within the reach of most research and industrial laboratories.

Water desorption in Kelvin-probe force microscopy: a generic model.

Nanoparticles or similar, nanoscale objects such as proteins or biological fibrils usually have to be deposited from aqueous suspension onto a solid support surface for further characterization by atomic force microscopy (AFM) and related methods such as Kelvin-probe force microscopy (KFM). Here we show, on the examples of functionalized nanoparticles and collagen fibrils, that water desorption after sample preparation affects their electrostatic potential determined by KFM in a predictable manner. We explain this effect with a simple, analytical model based on the capacitance of the partially dielectric-filled tip-sample system. We also propose practical measures to avoid false interpretation of electrical AFM-based experiments. As the phenomenon is very generic it may have significant implications in the application of AFM to nanoparticles and other nanostructures including biological ones.

Active damping by Q-control for fast force-distance curve measurements in atomic force microscopy.

This paper investigates the benefit of active damping by an analog Q-control circuit for measuring fast force-distance curves in atomic force microscopy. By active damping of the cantilever oscillation after snap-off, the down-ring time-constant is reduced significantly from 385 µs to 23 µs. Experimental results demonstrate that the number of force-distance curves per second can be increased by a factor of more than 30.

Survey of naturally and conventionally cured commercial frankfurters, ham, and bacon for physio-chemical characteristics that affect bacterial growth.

Natural and organic food regulations preclude the use of sodium nitrite/nitrate and other antimicrobials for processed meat products. Consequently, processors have begun to use natural nitrate/nitrite sources, such as celery juice/powder, sea salt, and turbinado sugar, to manufacture natural and organic products with cured meat characteristics but without sodium nitrite. The objective of this study was to compare physio-chemical characteristics that affect Clostridium perfringens and Listeria monocytogenes growth in naturally cured and traditionally cured commercial frankfurters, hams, and bacon. Correlations of specific product characteristics to pathogen growth varied between products and pathogens, though water activity, salt concentration, and product composition (moisture, protein and fat) were common intrinsic factors correlated to pathogen growth across products. Other frequently correlated traits were related to curing reactions such as % cured pigment. Residual nitrite and nitrate were significantly correlated to C. perfringens growth but only for the ham products.

Inhibition of Listeria monocytogenes using natural antimicrobials in no-nitrate-or-nitrite-added ham.

Consumer demand for foods manufactured without the direct addition of chemical preservatives, such as sodium nitrite and organic acid salts, has resulted in a unique class of "naturally" cured meat products. Formulation with a natural nitrate source and nitrate-reducing bacteria results in naturally cured processed meats that possess traits similar to conventionally cured meats. However, previous research has shown that the naturally cured products are more susceptible to pathogen growth. This study evaluated Listeria monocytogenes growth on ham manufactured with natural curing methods and with commercially available clean-label antimicrobials (cultured sugar and vinegar blend; lemon, cherry, and vinegar powder blend) and assessed impacts on physicochemical characteristics of the product. Hams made with either of the antimicrobials supported L. monocytogenes growth similar to that in the traditionally cured control (P > 0.05). Hams made with prefermented celery juice powder had the lowest residual nitrite concentrations (P < 0.05), and when no antimicrobial was added, L. monocytogenes growth was similar to that of the uncured control (P > 0.05). Aside from residual nitrite and nitrate concentrations, few physicochemical differences were identified. These findings show that ham can be produced with natural curing methods and antimicrobials to provide similar L. monocytogenes inhibition and physicochemical traits as in traditionally cured ham.

Mutations in the STAT5A gene are associated with embryonic survival and milk composition in cattle.

The objective of this study was to investigate the association of the signal transducer and activator of transcription 5A (STAT5A) gene with fertilization rate, embryonic survival, and milk production and composition in cattle. The STAT proteins are transcription factors that are specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. The STAT5A gene is a member of the interferon-tau (IFN-tau) and placental lactogen (PL) signaling pathway, which is involved in both milk production and initiation of pregnancy. Using the DNA-pooling sequencing approach, a total of 12 single nucleotide polymorphisms (SNP) were identified, 1 exonic and 11 intronic. For the study of association of these SNP with embryonic survival, 1,551 embryos were produced in vitro from 160 cows and 3 sires. Significant associations with embryonic survival were found for 7, 5, and 2 SNP for embryos produced from sires 1, 2, and 3 respectively. The association of fertilization rate with STAT5A polymorphisms was evaluated in more than 2,300 oocytes. Significant associations were found for 6, 2, and 2 SNP for sires 1, 2, and 3 respectively. For sire 1, 5 SNP showed significant associations with both embryonic survival and fertilization rate compared with 1 SNP for sires 2 and 3. To determine if embryonic losses had occurred before the blastocyst stage, 145 of the surviving embryos were harvested at d 7 of development and genotyped for the single exonic SNP12195. A significant segregation distortion was observed between oocytes produced from 2 sires carrying the same genotype. Thus, it is most likely that STAT5A is associated with 2 mechanisms of embryo death. One is a prefertilization mechanism involving sperm factors that cause low fertilization rate. The second is a postfertilization mechanism that causes incompatibility between the male pronucleus and the oocyte, which in turn leads to death of the embryo before the blastocyst stage. Association testing of SNP12195 (exon 8) and SNP14217 (intron 9) with milk composition revealed that allele G of SNP12195 was associated with a decrease in both protein and fat percentages. However, SNP14217 in intron 9 showed no significant association with milk production or health traits. The G allele of SNP12195 was also associated with low embryonic survival, making this SNP an attractive candidate for progeny testing programs in dairy cattle.

Continuous complete hematological and cytogenetic remission with molecular minimal residual disease 9 years after discontinuation of interferon-alpha in a patient with Philadelphia chromosome-positive chronic myeloid leukemia.

Interferon-alpha has been used as standard therapy for patients with Philadelphia-positive chronic myeloid leukemia (CML) for more than 20 years. Recently randomized trials have shown a superiority of the tyrosine kinase inhibitor imatinib in respect to its efficacy to induce complete hematological and cytogenetic remissions and more importantly in overall survival. Although follow-up is much shorter for imatinib than for interferon-alpha, this data changed the treatment algorithms in this disease. At the end of the era of interferon-alpha as a single-drug first-line treatment for most patients we present a case report which exemplifies a rare but exciting property of interferon-alpha in CML: the induction of complete hematological and cytogenetic remissions which can persist over years after discontinuation of the drug. Hence, the enrollment of CML patients in clinical trials which explore a combination treatment of imatinib and interferon-alpha is warranted.

[Endoscopic removal of an intraluminal duodenal diverticulum in an adult with upper abdominal pain]. / Endoskopische Spaltung eines intraluminalen Duodenaldivertikels beim Erwachsenen wegen Oberbauchbeschwerden.

We report here on a 44-year-old previously healthy patient with a two-year history of intermittent upper abdominal pain. In the outpatient gastroduodenoscopy and X- ray examinations of the small intestine an intraluminal duodenal diverticulum was suspected. Clinical examination and laboratory tests did not show any abnormal findings. In order to exclude other causes for the patient's complaints coloscopy, ERP and MRCP were performed. The latter was done because the bile duct could not be intubated in the ERCP due to the altered anatomy. By use of endoscopic ultrasound a mucosal duplication was demonstrated and thus the diagnosis confirmed. Subsequently, the diverticulum sac was sliced by argon plasma coagulation. The postinterventional course was without complications and the patient was without symptoms afterwards. The intraluminal duodenal diverticulum is a rare differential diagnosis of pain in the upper abdomen. The diverticulum should be endoscopically removed if other causes for abdominal pain have been ruled out and possibly associated malformations have been excluded.

Renal complications of high-dose chemotherapy and peripheral blood stem cell transplantation.

Following bone marrow transplantation, acute renal failure and proteinuria are common complications with a high mortality, particularly in patients requiring hemodialysis. Incidence, potential predisposing factors, and outcome of acute renal complications in patients with hematological malignancies receiving autologous peripheral blood stem cell transplantation were prospectively studied in 53 patients. Eight patients developed acute renal failure. Three of them required hemodialysis. Of all patients with acute renal failure, only those requiring hemodialysis died, due to nonrenal causes. Only 1 of the 45 patients without renal failure died. Mild proteinuria of predominantly tubular origin occurred in 16 patients, in 3 with and in 13 without acute renal failure. As predisposing factors for acute renal failure were identified: renal hypoperfusion due to systemic inflammatory response syndrome, sepsis or septic shock, and combined administration of nephrotoxic drugs. Especially those patients receiving high numbers of nephrotoxic drugs in combination with renal hypoperfusion were likely to develop acute renal failure. These results suggest that patients receiving high-dose chemotherapy and autologous peripheral blood stem cell transplantation have a low risk of developing acute renal failure and proteinuria.

Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains six structural proteins the roles of which are not completely understood. In a preceding study, immunization with the dutch isolate I10 of PRRSV had led to the development of MAbs against four structural proteins [Wieczorek-Krohmer, M., 1994. Herstellung und Charakterisierung von monoklonalen Antikörpern gegen das Virus des Porzinen Reproduktiven und Respiratorischen Syndroms (PRRSV). Inaugural-Dissertation, Ludwig-Maximilians-Universität, München] here finally identified by reaction with individual plasmid-expressed PRRSV proteins as products of ORFs 3 (GP3), 4 (GP4), 5 (GP5) and 7 (N). Surprisingly, the MAbs against GP5 revealed the presence of two antigenically distinct virus populations in the isolate I10, the population PRRSV-'PPV', isolated from plaques and the PRRSV-'EPV', gained by end point dilution. MAbs against GP3, GP4 and N reacted with both I10 populations as well as with natural PRRSV isolates. However, the anti-GP5 MAbs exclusively recognized PRRSV-'PPV'. In this study immunization of mice with both separated I10 populations confirmed that solely PRRSV-'PPV' possesses the property to induce an immune response ultimately leading to the establishment of MAbs against GP5. Whereas the 15 anti-GP5 MAbs (derived from four independent fusions) reacted exclusively with PRRSV-'PPV' of the isolate I10, anti-GP4 MAbs detected their target antigen on various isolates of European origin and were able to neutralize them. As indicated by competition assays and selection of neutralization-resistant virus mutants, all GP5 MAbs are directed against a single antigenic site on the ORF 5 protein. Both groups of neutralizing antibodies bound to the surface of purified virions demonstrating that the recognized epitopes represent surface structures of the virion envelope. However, anti-GP5 MAbs mediated the binding of more gold granules than anti-GP4 MAbs. Comparison of the neutralizing effect of anti-GP4 and anti-GP5 MAbs revealed the anti-GP5 MAbs as the more efficient antibodies. For the complete neutralization of about 100 ID50 of PRRSV-'PPV' anti-GP5 culture supernatant was effective up to a dilution of 1:1280 whereas the most effective anti-GP4 antibodies exhibited a comparable effect only up to 1:64. These results indicate that PRRSV GP5 in principle is a major target for neutralizing antibodies, as is found for other arteriviruses, but that in nature 'ORF 5 escape mutants' may develop as easily as in vitro.

Evidence for channeling of intermediates in the oxidative pentose phosphate pathway by soybean and pea nodule extracts, yeast extracts, and purified yeast enzymes.

Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next. CO2 produced from [1-14C]glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway-produced intermediates are in equilibrium with identical molecules in the bulk phase. This was the case whether glucose 6-phosphate (Glc6P), 6-phosphogluconolactone, or 6-phosphogluconate was added. Assumptions involved in calculating the amount of 14CO2 predicted for free mixing of 14C-labelled and unlabelled intermediates are discussed, together with the following results. (a) 14CO2 production by pea nodules in the presence of 3 mM 6-phosphogluconate was higher than in its absence. (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract. (c) 6-Phosphogluconate and 6-phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase despite the absence of 6-phosphogluconolactonase in the purified yeast enzyme mixture. (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling. (e) Poly(ethylene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes. On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes. Taken together, the results support channeling of OPPP pathway intermediates.

Anti-HIV-1 activity of inorganic polyphosphates.

Human blood plasma, serum, peripheral blood mononuclear cells, and erythrocytes contain significant amounts of inorganic polyphosphates (ranging from 53 to 116 microM, in terms of phosphate residues). Here we demonstrate that at higher concentrations linear polyphosphates display cytoprotective and antiviral activity. Sodium tetrapolyphosphate and the longer polymers, with average chain lengths of 15, 34, and 91 phosphate residues, significantly inhibited human immunodeficiency virus type 1 (HIV-1) infection of cells in vitro at concentrations > or = 33.3 microg/ml (> or = 283-324 microM phosphate residues), whereas sodium tripolyphosphate was ineffective. In the tested concentration range, these compounds had no effect on cell growth. The longer-chain polyphosphates (polyphosphates with mean chain lengths of 15 and 34) but not sodium tripolyphosphate and sodium tetrapolyphosphate also inhibited HIV-1-induced syncytium formation at a concentration of 160 microg/ml (1.51-1.54 mM phosphate residues). The results obtained with the syncytium assay and by cell-virus binding experiments indicate that the anti-HIV effect of these nontoxic polyanions may be caused by binding of the compounds to both the host cell surface and the virus, thereby inhibiting adsorption of the virus. Competition experiments revealed that binding of [32P]polyphosphate to Molt-3 cells was only partially inhibited by the antibody OKT4A.

Porcine reproductive and respiratory syndrome virus (PRRSV): monoclonal antibodies detect common epitopes on two viral proteins of European and U.S. isolates.

Sixteen hybridoma cell lines secreting monoclonal antibodies (mAbs) directed against two dutch isolates of the causative virus of Porcine Reproductive and Respiratory Syndrome (PRRSV) were produced. The hybridoma cells resulted from fusions of SP2/0 myeloma cells with splenocytes of STU mice immunized with purified PRRSV after induction of immunotolerance against host cell constituents. Screening of supernatant fluids was performed by an indirect immunofluorescence assay on PRRSV-infected porcine alveolar macrophages. Immunoblotting studies revealed that the mAbs had different protein specificities. One mAb reacted with a viral 15 kD protein, eleven were directed against a 40-50 kD protein, and four against a 30-40 kD protein. The mAb against the 15 kD putative nucleocapsid protein as well as five mAbs against the 40-50 kD protein recognized epitopes on these proteins which are conserved in various European and U.S. isolates of PRRSV.

Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum.

We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses.

Team treatment of eating disorders.

The treatment of eating disorders requires the expertise of a multidisciplinary team. This case presents the psychologic and physiologic challenges observed in managing these patients. Attention must be directed toward the use of techniques to obtain a commitment from patients to meet a therapeutic goal. Care must be taken to avoid overaggressive feeding of patients with eating disorders to avoid the refeeding syndrome.

The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.

Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, delta 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteriods dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps delta 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.