Detection of HIV-1 in alternative specimen types using the APTIMA HIV-1 RNA Qualitative Assay.

Peripheral blood mononuclear cells (PBMCs), saliva, seminal plasma, and dried blood spots were evaluated as specimen types for the APTIMA HIV-1 RNA Qualitative Assay (APTIMA HIV-1 Assay), which employs a target capture step to recover HIV-1-specific sequences from complex specimen types. Analytical sensitivity studies were carried out using samples that were either diluted or eluted with a buffered detergent and spiked with different concentrations of HIV-1 ranging from 1 to 10,000 copies/mL. PBMC samples spiked with HIV-1 had comparable analytical sensitivity to HIV-1 spiked plasma with a 95% limit of detection of 13.1 and 17.2 copies/mL, respectively. Analytical sensitivity in seminal plasma specimens diluted 1:5 and saliva diluted 1:2 was comparable to HIV-1 spiked dilution buffer alone. Whole blood and dried blood spot specimens spiked with HIV-1 had equivalent reactivity at 250 copies/spot (5000 copies/mL). However, the 95% limit of detection values were significantly different (293.7 copies/mL for whole blood and 2384 copies/mL for dried blood spot specimens). No significant effect on analytical sensitivity was observed when one HIV-1 positive dried blood spot punch was pooled with up to 9 HIV-1 negative dried blood spot punches. Together, these studies demonstrate that the APTIMA HIV-1 RNA Qualitative Assay can be used to process a diverse array of specimen types with minimal impact on analytical sensitivity for most specimen types.

Anti-VEGF antibody bevacizumab (Avastin) with 5FU/LV as third line treatment for colorectal cancer.

BACKGROUND: To evaluate the activity and safety of bevacizumab when given with standard 5FU/leukovorin (LV) regimens in patients with metastatic colorectal cancer who have failed irinotecan and oxaliplatin-based treatments. METHODS: Bevacizumab was given at 5 mg/kg as an IV infusion every 2 weeks. Patients received 5FU according to Roswell Park or the de Gramont regimen. RESULTS: Nineteen patients enrolled, median age 60, median PS: 1. Most common toxicity attributable to bevacizumab was mild hypertension, epistaxis and mild proteinuria; 1 patient had a CNS haemorrhage. The median number of cycles was 1 (8 weeks). Clinical benefit as disease stabilisation lasting 2-6 months was noted in 9 patients, whereas 10 progressed (median f/u: 5 months). TTP was 16 weeks, and the overall survival has not been reached (24+ weeks). CONCLUSIONS: Bevacizumab may result in growth arrest and clinical benefit in a substantial proportion of patients with colorectal cancer and no alternative treatment.

[Waiting for the EuGH verdict to be put into practice or "Introduction of the four-day week on full pay"]. / Warten auf die Umsetzung des EuGH-Urteils oder "Einführung der Viertagewoche bei vollem Lohnausgleich"

The submitted model of working time transposes and interprets german industrial law. The result of this interpretation is a high level of acceptance of the employees, a fast education that is high qualified with costs that are still affordable. The advantage of this model compared with the shift-model that runs after the EuGH-decision is obvious if you look at the reality of our health care system. This is why it is important to have an efficient interpretation of the existing law. Of course it will be a necessity also in the future to create new models of working time and to adapt these models in a way that it fits into the structure of a hospital. It would be the wrong way to force a juridical and political decision, how it was done by the german government that gave a deadline to put the EuGH decision into operation, without the possibility of an interpretation that fulfils the demand of the hospital.

Cell death during development of intercalated ducts in the rat submandibular gland.

Programmed cell death, or apoptosis, occurs during the development of many tissues and organs in almost all multicellular organisms. Although apoptosis of salivary gland cells has been demonstrated in several pathological conditions, the role of apoptosis in the postnatal development of the salivary glands is unknown. We have studied the development of the rat submandibular gland (SMG) during its transition from the perinatal stage to the mature adult stage. Terminal tubule or Type I cells, which synthesize the secretory protein SMG-C, are prominent in the perinatal acini and are believed to form the intercalated ducts of the adult gland. Between 25 days and 30 days after birth, the number of Type I cells and their SMG-C immunoreactivity markedly decreased. Apoptotic cells in association with the developing intercalated ducts were labeled with the Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) method. Between 25 and 40 days of age, from 50 to 80% of the apoptotic cells in cryostat sections of the SMG were closely associated with the intercalated ducts. Electron microscopy showed that the Type I cells became vacuolated, their secretory granules were reduced in size and number, and the amount of rough endoplasmic reticulum was decreased. Cellular debris resembling apoptotic bodies was phagocytosed by macrophages and adjacent intercalated duct cells. These observations suggest that the loss of Type I cells and reduction of SMG-C immunoreactivity during development of the intercalated ducts of the adult rat SMG is due, at least in part, to apoptosis.

Efficacy of exogenous recombinant murine leptin in lean and obese 10- to 12-mo-old female CD-1 mice.

Leptin efficacy was compared in obese and lean female CD-1 mice. Body weights in these 10- to 12-mo-old mice ranged from 29.7 to 62.0 g, and leptin levels correlated with body weight. Mice from the lean and obese ends of the weight distribution were treated with daily peripheral leptin injections (1-100 mg/kg) for a 33-day period. The half-maximal effective doses for weight loss and fat reduction were shifted 0.5-0.7 log to the right for obese mice. Leptin was less efficacious at low doses (1-3 mg/kg) in obese mice but equal to or more efficacious in obese than lean mice at high doses (30-100 mg/kg). Leptin's initial effects on weight loss could be explained by appetite suppression in both groups, but its effects on fat reduction were greater in leptin-treated than pair-fed mice, particularly in the lean group. Leptin also prevented the elevations in serum corticosterone and ketones found in pair-fed lean mice. These data allow a quantitative comparison of leptin sensitivity in obese vs. lean CD-1 mice and suggest that in mice where obesity is a function of outbreeding and age, leptin sensitivity is moderately reduced. Furthermore, although appetite suppression has a clear role in leptin's effects on body weight, leptin may also have specific effects on lipid metabolism and mobilization that are different from the metabolic compensations that normally occur with food deprivation.

Sedation in children: adequacy of two-hour fasting.

OBJECTIVES: (1) To investigate the relationship between the duration of time that children fasted before a procedure and their gastric volume and pH at the time of the procedure. (2) To compare the variables of gastric pH and volume with historical standards. METHODS: We performed 285 gastroscopies for children aged 0.1 to 18.6 years (mean, 7.5 +/- 5.3) between October 1991 and January 1995. Duration of fasting was 0.5 to 24 hours (mean, 6.7 +/- 5.3) after ingestion of clear liquids. Immediately after intravenously administered sedation, the gastric contents were removed endoscopically with suction and direct visualization to ensure complete evacuation. The volume and pH of the gastric contents were measured and analyzed in comparison with the duration of fasting. The values obtained were also compared with historical standards thought to minimize the risk of aspiration pneumonia: gastric volume 0.4 ml or less per kilogram of body weight and pH of 2.5 or greater. RESULTS: There was no significant correlation between duration of fasting and either gastric volume divided by body weight (mean, 0.68 +/- 1.31 ml/kg; range, 0 to 15.23 ml/kg) or pH (mean, 2.03 +/- 1.40; range, 1 to 8). There was less no significant difference in the percentage of children with gastric volume of 0.4 ml/kg or less or with pH of 2.5 or greater between the groups with the following fasting times: 30 minutes to 3 hours, more than 3 hours to 8 hours, and more than 8 hours. CONCLUSIONS: On the basis of the data in this study and a review of the literature, we concluded that (1) fasting longer than 2 hours after ingesting clear liquids does not significantly change gastric volume or pH, (2) there is no advantage in requiring children to fast for longer than 2 hours after clear liquid ingestion before sedation or anesthesia for any procedure, and (3) fewer than half of pediatric patients actually achieve the "desirable" values of a gastric volume of 0.4 ml/kg or less and a pH value of 2.5 pH units or more, regardless of fast duration, even though these values are presented in the literature as a goal to minimize the risk of aspiration pneumonia.

Autocatalytic networks with intermediates. I: Irreversible reactions.

A class of autocatalytic reaction networks based on template-dependent replication and specific catalysis is considered. Trimolecular "elementary steps" of simple replicator dynamics are resolved into two consecutive irreversible reactions. The extreme cases, competition for common resources and hypercycle-like cooperative feedback, were analyzed in some detail. Although the dynamics of the extended networks resembles corresponding replicator dynamics in general, there are significant differences. Most notably, the interior fixed points in the cooperative model turned out to be asymptotically stable for an arbitrary number of species, whereas simple replicator dynamic predicts an asymptotically stable periodic orbit fixed for four species and fewer and a stable periodic orbit for all other cases.

An improved intravital microscopy system.

The use of intravital microscopy as a tool for studying the microcirculation has increased greatly over the last several decades. Early microscopes provided the first pictures of the microcirculation, but were cumbersome to use and subjected the tissue to a high light intensity, a problem which has recently become the subject of much discussion. The goal of this project was therefore to build a more ergodynamic microscope which minimizes the light exposure to the tissue. The automation of the microscope controls provides a platform on which other options can be built into the microscope, such as an autofocus feature. Furthermore, the use of the Optimas software also opens the possibility for on-line data processing.

Refined crystal structure and mutagenesis of human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) has been refined against data extending to a resolution of approximately 2.4 A along a* and approximately 1.9 A along b* and c*. Anisotropic scale factors of B11 = -20.8 A2, B22 = 7.4 A2, B33 = 13.3 A2 corrected for the more rapid fall of diffraction in the a* direction. The anisotropy correlates with the weak crystal packing interactions along the a axis. In addition to apolar side chains in the protein core, there are 10 buried hydrogen bonding residues. Those residues involved in intramolecular hydrogen bonding to main chain atoms are better conserved than those hydrogen bonding to other side chain atoms; 24 solvation sites are observed at equivalent positions in the two molecules in the asymmetric unit, and the strongest among these are located in clefts between secondary structural elements. No buried water sites are seen. Two surface clusters of hydrophobic side chains are located near the expected receptor binding regions. Mutagenesis of 11 residues on the helix A/helix C face confirms the importance of Glu-21 and shows that Gly-75 and Gln-86, located on helix C, each cause a greater than fourfold drop in activity. Glu-21 and Gly-75, but not Gln-86, are structurally equivalent to residues involved in the growth hormone binding to its receptor.

Preparative electrofocusing for industrial applications.

This paper presents a case study of using a multicompartment isoelectric focusing apparatus to determine the isoelectric points and focus preparative quantities of brain derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3). A separation of PEGylated from unPEGylated forms of granulocyte colony stimulating factor (G-CSF) is described as well. Both BDNF and NT3 have substantially higher pI values in this system than is predicted from sequenced based modeling. Although PEGylated forms of G-CSF can be separated from the unPEGylated forms, separation of protein with differing degrees of PEGylation was not achieved. The paper additionally demonstrates that this technique can be used simultaneously as an analytical and preparative tool, eliminating the need for analytical IEF gels.

A novel set of uncoordinated mutants in Caenorhabditis elegans uncovered by cold-sensitive mutations.

A set of uncoordinated (Unc) cold-sensitive (cs) mutants was isolated at a stringent condition of 11 degrees C. About half of the 13 independently isolated cs-Unc mutants were alleles of three X-linked Unc mutants that exhibited the "kinker" phenotype. The remaining four isolates identified new mutants that exhibited "kinker," "coiler," or severe paralytic phenotypes. The temperature-sensitive period (TSP) for each gene was determined. As a homozygous or heterozygous dominant, unc-125 exhibited a TSP throughout all stages of development. Its severe paralysis was immediately observed upon a shift down to 11 degrees C and reversed upon a shift up to 23 degrees C. The reversible thermolability of the unc-125 gene product indicated that it may function in a multicomponent process involved in neuro-excitation.

Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceraldehyde-3-phosphate dehydrogenase at 25 Angstroms Resolution.

The crystal structure of holo D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophile Thermus aquaticus has been solved at 2.5 Angstroms resolution. To study the determinants of thermostability, we compare our structure to four other GAPDHs. Salt links, hydrogen bonds, buried surface area, packing density, surface to volume ratio, and stabilization of alpha-helices and beta-turns are analyzed. We find a strong correlation between thermostability and the number of hydrogen bonds between charged side chains and neutral partners. These charged-neutral hydrogen bonds provide electrostatic stabilization without the heavy desolvation penalty of salt links. The stability of thermophilic GAPDHs is also correlated with the number of intrasubunit salt links and total hydrogen bonds. Charged residues, therefore, play a dual role in stabilization by participating not only in salt links but also in hydrogen bonds with a neutral partner. Hydrophobic effects allow for discrimination between thermophiles and psychrophiles, but not within the GAPDH thermophiles. There is, however, an association between thermostability and decreasing enzyme surface to volume ratio. Finally, we describe several interactions present in both our GAPDH and a hyperthermophilic GAPDH that are absent in the less thermostable GAPDHs. These include a four-residue salt link network, a hydrogen bond near the active site, an intersubunit salt link, and several buried Ile residues.

Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice.

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.

Megakaryocyte growth and development factor and interleukin-3 induce patterns of protein-tyrosine phosphorylation that correlate with dominant differentiation over proliferation of mpl-transfected 32D cells.

Recently, the ligand for c-mpl, a member of the family of cytokine receptors, was cloned and found to be a physiologic regulator of platelet homeostasis. We report that megakaryocyte growth and development factor (MGDF, thrombopoietin [TPO], c-mpl ligand ) induces differentiation in a majority of mpl-transfected 32D cells, while interleukin (IL)-3 is exclusively mitogenic in this system. MGDF differentiation, as measured by decreased proliferation, changes in cellular morphology, increased adherence, and downregulation of very late antigen (VLA)-4, is dominant over IL-3 proliferation. MGDF induces tyrosine-phosphorylation of mpl, JAK2, SHC, SHPTP-1 (HCP, motheaten) and SHPTP-2 (Syp, PTP-1D) within 30 seconds of stimulation, as well as of vav and MAPK with slightly delayed kinetics. A fraction of mpl and JAK2 is preassociated, and the stoichiometry of this complex is unaltered by cytokine stimulation. After MGDF stimulation, we detect interactions among SHC, grb2, SHPTP-1, SHPTP-2, and the mpl/JAK2 complex. IL-3 induces phosphorylation of the above proteins with the exception of mpl and also causes weak JAK1 phosphorylation. Although similar in composition, the MGDF- and IL-3-induced complexes of signal transducers appear to be assembled in different configurations, especially with respect to SHPTP-2. Both MGDF and IL-3 induce tyrosine phosphorylation of STAT3 (APRF) and STAT5 (MGF), with MGDF favoring STAT3 while IL-3 predominantly causes STAT5 phosphorylation. In addition, some proteins become tyrosine-phosphorylated in response to MGDF only, suggesting that we may have detected differentiation-specific signal transducers. These include a number of high-molecular-weight proteins (140 to 200 kD) and one 28-kD protein that becomes tyrosine-phosphorylated only briefly.

Effects of the obese gene product on body weight regulation in ob/ob mice.

C57BL/6J mice with a mutation in the obese (ob) gene are obese, diabetic, and exhibit reduced activity, metabolism, and body temperature. Daily intraperitoneal injection of these mice with recombinant OB protein lowered their body weight, percent body fat, food intake, and serum concentrations of glucose and insulin. In addition, metabolic rate, body temperature, and activity levels were increased by this treatment. None of these parameters was altered beyond the level observed in lean controls, suggesting that the OB protein normalized the metabolic status of the ob/ob mice. Lean animals injected with OB protein maintained a smaller weight loss throughout the 28-day study and showed no changes in any of the metabolic parameters. These data suggest that the OB protein regulates body weight and fat deposition through effects on metabolism and appetite.

[A new method for measuring internal ear circulation using colored microspheres]. / Eine neue Methode zur Messung der Innenohrdurchblutung mit Hilfe farbiger Mikrosphären.

The pathogenesis of sudden hearing loss is as yet unknown. Vascular disturbances are thought to play a major role, but they cannot be diagnosed clinically. We have therefore developed an animal model which allows measurements of the cochlear blood flow (CBF) and function. For CBF measurements a new technique based on coloured microspheres was developed. New Zealand rabbits were anaesthetised, ventilated and blood pressure and heart frequency were continuously monitored. 6 x 10(6) microspheres of one colour were injected in the left ventricle; different colours are available and allow multiple measurements. The amount of microspheres trapped in the capillary bed of the cochlea depends on the regional blood flow. An arterial reference blood sample makes it possible to calculate absolute values of the regional blood flow in the cochlea. The amount of microspheres trapped in the cochlea was determined microscopically after dissecting, dissolving and filtrating the cochlea. Measurements of the CBF in a control group (5 rabbits, 10 inner ears) at the beginning and after 120 minutes were performed. Mean CBF was 3.8 +/- 1.1 microliters/min and 4.3 +/- 1.3 microliters/min. The coloured microsphere technique allows repeated measurements of the CBF with good reproducibility and an acceptable standard deviation. CBF can be measured in absolute values, which is of advantage for determining a lower limit of the CBF for the cochlea function.

Characterization of the cDNA and three processed pseudogenes from the murine protein kinase CK2 alpha subunit.

Seven protein kinase CK2 alpha clones were isolated from a murine genomic DNA library. They were assigned to four different genomic loci (A,B,C,D). Locus D was previously identified as a processed pseudogene (Boldyreff et al. (1992) Biochem. Biophys. Res. Commun. 186, 723-730). Here we present sequences of genomic loci B and C and the murine CK2 alpha cDNA. Loci B and C are like locus D processed pseudogenes, however, with considerable differences among each other and to the cDNA, especially with respect to the lengths of the putative gene products. Genomic locus D would code for a protein of 82 amino acids, locus B for a protein of 132 amino acids and locus C product for the full length product of 391 amino acids as the murine cDNA.