Determination of lethality rate constants and D-values for Bacillus atrophaeus (ATCC 9372) spores exposed to dry heat from 115 degrees C to 170 degrees C.

Dry heat microbial reduction is the NASA-approved sterilization method to reduce the microbial bioburden on spaceflight hardware for missions with planetary protection requirements. The method involves heating the spaceflight hardware to temperatures between 104 degrees C and 125 degrees C for up to 50 hours, while controlling the humidity to very low values. Collection of lethality data at temperatures above 125 degrees C and with ambient (uncontrolled) humidity conditions would establish whether any microbial reduction credit can be offered to the flight project for processes that occur at temperatures greater than 125 degrees C. The goal of this research is to determine the survival rates of Bacillus atrophaeus (ATCC 9372) spores subjected to temperatures higher than 125 degrees C under both dry (controlled) and room ambient humidity (36-66% relative humidity) conditions. Spores were deposited inside thin, stainless steel thermal spore exposure vessels (TSEVs) and heated under ambient or controlled humidity conditions from 115 degrees C to 170 degrees C. After the exposures, the TSEVs were cooled rapidly, and the spores were recovered and plated. Survivor ratios, lethality rate constants, and D-values were calculated at each temperature. At 115 degrees C and 125 degrees C, the controlled humidity lethality rate constant was faster than the ambient humidity lethality rate constant. At 135 degrees C, the ambient and controlled humidity lethality rate constants were statistically identical. At 150 degrees C and 170 degrees C, the ambient humidity lethality rate constant was slightly faster than the controlled humidity lethality rate constant. These results provide evidence for possibly modifying the NASA dry heat microbial reduction specification.

Higher urinary excretion of essential amino acids in preterm infants fed protein hydrolysates.

AIM: Protein hydrolysates have been introduced in preterm formulae, but it is not clear whether they are needed for the feeding of preterm infants. We designed a randomized, controlled trial to test the effects of a preterm formula with hydrolysed cow's milk proteins on short-term growth and urinary and plasma amino acids levels. METHODS: Infants with a birthweight < or = 1750 g and gestational age < or = 34 wk fed a conventional preterm infant formula (formula B) or a hydrolysed formula (formula A). Weight was measured daily; length, head circumference, mid-arm circumference and total skinfold thickness were measured weekly. Blood and urine were analysed for amino acid concentrations at start, 14 and 28 d. RESULTS: Twenty-one infants met the criteria for randomization. The daily feeding volumes were: formula A 172.8 +/- 5.6 vs formula B 170.1 +/- 2.8 ml/kg/d. Infants fed with formula A showed slower weight gain (17.4 +/- 3.4 vs 20.5 +/- 3.3 g/kg/d; p = 0.045) and lower mean change in Z-scores for weight (-0.18 +/- 0.16 vs 0.00 +/- 0.09; p = 0.009) and for head circumference (-0.06 +/- 0.13 vs 0.06 +/- 0.13; p = 0.049). After 14 d, infants receiving formula A had statistically significant higher urinary levels of essential amino acids compared to infants receiving formula B. CONCLUSION: Our results support the hypothesis of less nutritional value of hydrolysed versus conventional preterm formulae. Higher renal excretion of essential amino acids may be one of the mechanisms involved. These findings must be confirmed by further studies with larger sample sizes and protein hydrolysates with different degrees of hydrolysis.

Structure and function of glutamyl-tRNA reductase involved in 5-aminolaevulinic acid formation.

In most bacteria, in archaea and in plants, the general precursor of all tetrapyrroles, 5-aminolaevulinic acid, is formed by two enzymes. The initial substrate, glutamyl-tRNA, is reduced by NADPH-dependent glutamyl-tRNA reductase to form glutamate 1-semialdehyde. The aldehyde is subsequently transaminated by glutamate-1-semialdehyde 2,1-aminomutase to yield 5-aminolaevulinic acid. The enzymic mechanism and the solved crystal structure of Methanopyrrus kandleri glutamyl-tRNA reductase are described. A pathway for metabolic channelling of the reactive aldehyde between glutamyl-tRNA reductase and the aminomutase is proposed.

V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis.

Processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls. tRNA-dependent catalysis generally is associated with protein biosynthesis. An exception is the reduction of glutamyl-tRNA to glutamate-1-semialdehyde by the enzyme glutamyl-tRNA reductase. This reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes. The crystal structure of glutamyl-tRNA reductase from the archaeon Methanopyrus kandleri in complex with the substrate-like inhibitor glutamycin at 1.9 A resolution reveals an extended yet planar V-shaped dimer. The well defined interactions of the inhibitor with the active site support a thioester-mediated reduction process. Modeling the glutamyl-tRNA onto each monomer reveals an extensive protein-tRNA interface. We furthermore propose a model whereby the large void of glutamyl-tRNA reductase is occupied by glutamate-1-semialdehyde-1,2-mutase, the subsequent enzyme of this pathway, allowing for the efficient synthesis of 5-aminolevulinic acid, the common precursor of all tetrapyrroles.

Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo.

The role of endothelial cell caveolae in the uptake and transport of macromolecules from the blood-space to the tissue-space remains controversial. To address this issue directly, we employed caveolin-1 gene knock-out mice that lack caveolin-1 protein expression and caveolae organelles. Here, we show that endothelial cell caveolae are required for the efficient uptake and transport of a known caveolar ligand, i.e. albumin, in vivo. Caveolin-1-null mice were perfused with 5-nm gold-conjugated albumin, and its uptake was followed by transmission electron microscopy. Our results indicate that gold-conjugated albumin is not endocytosed by Cav-1-deficient lung endothelial cells and remains in the blood vessel lumen; in contrast, gold-conjugated albumin was concentrated and internalized by lung endothelial cell caveolae in wild-type mice, as expected. To quantitate this defect in uptake, we next studied the endocytosis of radioiodinated albumin using aortic ring segments from wild-type and Cav-1-null mice. Interestingly, little or no uptake of radioiodinated albumin was observed in the aortic segments from Cav-1-deficient mice, whereas aortic segments from wild-type mice showed robust uptake that was time- and temperature-dependent and competed by unlabeled albumin. We conclude that endothelial cell caveolae are required for the efficient uptake and transport of albumin from the blood to the interstitium.

Structure of Urtica dioica agglutinin isolectin I: dimer formation mediated by two zinc ions bound at the sugar-binding site.

Ultica dioica agglutinin, a plant lectin from the stinging nettle, consists of a total of seven individual isolectins. One of these structures, isolectin I, was determined at 1.9 A resolution by the X-ray method. The crystals belong to the space group P2(1) and the asymmetric unit contains two molecules related by local twofold symmetry. The molecule consists of two hevein-like chitin-binding domains lacking distinct secondary structure, but four disulfide bonds in each domain maintain the tertiary structure. The backbone structure of the two independent molecules is essentially identical and this is similarly true of the sugar-binding sites. In the crystal, the C-terminal domains bind Zn(2+) ions at the sugar-binding site. Owing to their location near a pseudo-twofold axis, the two zinc ions link the two independent molecules in a tail-to-tail arrangement: thus, His47 of molecule 1 and His67 of molecule 2 coordinate the first zinc ion, while the second zinc ion links Asp75 of molecule 1 and His47 of molecule 2.

Identification and characterization of a cell surface marker for embryonic rat spinal accessory motor neurons.

The developing mammalian spinal cord contains distinct populations of motor neurons that can be distinguished by their cell body positions, by the expression of specific combinations of regulatory genes, and by the paths that their axons take to exit the central nervous system (CNS). Subclasses of spinal motor neurons are also thought to express specific cell surface proteins that function as receptors which control the guidance of their axons. We identified monoclonal antibody (mAb) SAC1 in a screen aimed at generating markers for specific subsets of neurons/axons in the developing rat spinal cord. During early embryogenesis, mAb SAC1 selectively labels a small subset of Isl1-positive motor neurons located exclusively within cervical segments of the spinal cord. Strikingly, these neurons extend mAb SAC1-positive axons along a dorsally directed trajectory toward the lateral exit points. Consistent with the finding that mAb SAC1 also labels spinal accessory nerves, these observations identify mAb SAC1 as a specific marker of spinal accessory motor neurons/axons. During later stages of embryogenesis, mAb SAC1 is transiently expressed on both dorsally and ventrally projecting spinal motor neurons/axons. Interestingly, mAb SAC1 also labels the notochord and floor plate during most stages of spinal cord development. The mAb SAC1 antigen is a 100-kD glycoprotein that is likely to be the rat homolog of SC1/BEN/DM-GRASP, a homophilic adhesion molecule that mediates axon outgrowth and fasciculation.

Internalins from the human pathogen Listeria monocytogenes combine three distinct folds into a contiguous internalin domain.

Listeria monocytogenes is an opportunistic, food-borne human and animal pathogen. Host cell invasion requires the action of the internalins A (InlA) and B (InlB), which are members of a family of listerial cell-surface proteins. Common to these proteins are three distinctive N-terminal domains that have been shown to direct host cell-specific invasion for InlA and InlB. Here, we present the high-resolution crystal structures of these domains present in InlB and InlH, and show that they constitute a single "internalin domain". In this internalin domain, a central LRR region is flanked contiguously by a truncated EF-hand-like cap and an immunoglobulin (Ig)-like fold. The extended beta-sheet, resulting from the distinctive fusion of the LRR and the Ig-like folds, constitutes an adaptable concave interaction surface, which we propose is responsible for the specific recognition of the host cellular binding partners during infection.

The N-terminal domain of Homer/Vesl is a new class II EVH1 domain.

Cellular activities controlled by signal transduction processes such as cell motility and cell growth depend on the tightly regulated assembly of multiprotein complexes. Adapter proteins that specifically interact with their target proteins are key components required for the formation of these assemblies. Ena/VASP-homology 1 (EVH1) domains are small constituents of large modular proteins involved in microfilament assembly that specifically recognize proline-rich regions. EVH1 domain-containing proteins are present in neuronal cells, like the Homer/Vesl protein family that is involved in memory-generating processes. Here, we describe the crystal structure of the murine EVH1 domain of Vesl 2 at 2.2 A resolution. The small globular protein consists of a seven-stranded antiparallel beta-barrel with a C-terminal alpha-helix packing alongside the barrel. A shallow groove running parallel with beta-strand VI forms an extended peptide-binding site. Using peptide library screenings, we present data that demonstrate the high affinity of the Vesl 2 EVH1 domain towards peptide sequences containing a proline-rich core sequence (PPSPF) that requires additional charged amino acid residues on either side for specific binding. Our functional data, substantiated by structural data, demonstrate that the ligand-binding of the Vesl EVH1 domain differs from the interaction characteristics of the previously examined EVH1 domains of the Evl/Mena proteins. Analogous to the Src homology 3 (SH3) domains that bind their cognate ligands in two distinct directions, we therefore propose the existence of two distinct classes of EVH1 domains.

Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.

A neural classifier enabling high-throughput topological analysis of lymphocytes in tissue sections.

A neural cell detection system (NCDS) for the automatic quantitation of fluorescent lymphocytes in tissue sections is presented in this paper. The system acquires visual knowledge from a set of training cell-image patches selected by a user. The trained system evaluates an image in 2 min calculating: the number, the positions, and the phenotypes of the fluorescent cells. For validation, the NCDS learning performance was tested by cross validation on digitized images of tissue sections obtained from inherently different types of tissue: diagnostic tissue sections across the human tonsil and across an inflammatory lymphocyte infiltrate of the human skeletal muscle. The NCDS detection results were compared with detection results from biomedical experts and were visually evaluated by our most experienced biomedical expert. Although the micrographs were noisy and the fluorescent cells varied in shape and size, the NCDS detected a minimum of 95% of the cells. In contrast, the cellular counts based on visual cell recognition of the experts were inconsistent and largely unreproducible for approximately 80% of the lymphocytes present in a visual field. The data indicate that the NCDS is rapid and delivers highly reproducible results and, therefore, enables high-throughput topological screening of lymphocytes in many types of tissue, e.g., as obtained by routine diagnostic biopsy procedures. High-throughput screening with the NCDS provides the platform for the quantitative analysis of the interrelationship between tissue environment, cellular phenotype, and cellular topology.

The distally based radial forearm fascia-fat flap for treatment of recurrent de Quervain's tendonitis.

A radial fascial flap has been described as a valuable and versatile option to provide appropriate tissue coverage of tendons, nerves, and soft tissues in the forearm and hand. We report the use of this distally based radial forearm fascia-fat flap to create a fascial tube to treat recurrent de Quervain's tendonitis.

Prospective comparison of panoramic tomography (zonography) and helical computed tomography in the diagnosis and operative management of mandibular fractures.

This prospective study compared the sensitivity of panoramic tomography (zonography) and helical computed tomography (CT) in diagnosing 73 mandibular fractures in 42 consecutive patients and correlated the results with known surgical findings. The purpose of the study was to determine the optimal radiologic examination for the diagnosis and operative management of mandibular fractures. The attending surgeons' interpretations of panoramic tomograms and helical CT images in the axial plane were compared with the patients' known surgical findings. A series of questions assessed the relative contribution of these two radiologic examinations in formulating an optimal operative plan for each patient. In the 42 patients studied, the sensitivity of helical CT was 100 percent in diagnosing mandibular fractures; this compared with 86 percent (36 of 42) for panoramic tomography, in which significantly more fractures were missed (p = 0.0412). In the six patients with fractures not visualized, the operative management was altered because of the new fracture visualized on helical CT. Of the seven missed fractures, six were in the posterior portion of the mandible. Comparing fracture detection by region, seven fractures found on helical CT were not visualized on panoramic tomography. Helical CT improved the understanding of the nature of mandibular fractures by providing additional information regarding fracture displacement and comminution and by locating injuries missed using panoramic tomography. This study suggests that helical CT alone may be more diagnostic than panoramic tomography alone in evaluating mandibular fractures. Helical CT sufficiently demonstrated details of fractures in 41 of 42 patients; in one patient, the nature of a dental root fracture was better delineated by panoramic tomography.

A comparison of mutations induced by accelerated iron particles versus those induced by low earth orbit space radiation in the FEM-3 gene of Caenorhabditis elegans.

The fem-3 gene of Caenorhabditis elegans was employed to determine the mutation frequency as well as the nature of mutations induced by low earth orbit space radiation ambient to Space Shuttle flight STS-76. Recovered mutations were compared to those induced by accelerated iron ions generated by the AGS synchrotron accelerator at Brookhaven National Laboratory. For logistical reasons, dauer larvae were prepared at TCU, transported to either Kennedy Space Center or Brookhaven National Laboratory, flown in space or irradiated, returned to TCU and screened for mutants. A total of 25 fem-3 mutants were recovered after the shuttle flight and yielded a mutation frequency of 2.1x10(-5), roughly 3.3-fold higher than the spontaneous rate of 6.3x10(-6). Four of the mutations were homozygous inviable, suggesting that they were large deletions encompassing fem-3 as well as neighboring, essential genes. Southern blot analyses revealed that one of the 25 contained a polymorphism in fem-3, further evidence that space radiation can induce deletions. While no polymorphisms were detected among the iron ion-induced mutations, three of the 15 mutants were homozygous inviable, which is in keeping with previous observations that high LET iron particles generate deficiencies. These data provide evidence, albeit indirect, that an important mutagenic component of ambient space radiation is high LET charged particles such as iron ions.

Particle size distribution in effluent of trickling filters and in humus tanks.

Particles and aggregates from trickling filters must be eliminated from wastewater. Usually this happens through sedimentation in humus tanks. Investigations to characterize these solids by way of particle size measurements, image analysis and particle charge measurements (zeta potential) are made within the scope of Research Center for Science and Technology "Fundamentals of Aerobic biological wastewater treatment" (SFB 411). The particle size measuring results given within this report were obtained at the Ingolstadt wastewater treatment plant, Germany, which served as an example. They have been confirmed by similar results from other facilities. Particles flushed out from trickling filters will be partially destroyed on their way to the humus tank. A large amount of small particles is to be found there. On average 90% of the particles are smaller than 30 microm. Particle size plays a decisive role in the sedimentation behaviour of solids. Small particles need sedimentation times that cannot be provided in settling tanks. As a result they cause turbidity in the final effluent. Therefore quality of sewage discharge suffers, and there are hardly advantages of the fixed film reactor treatment compared to the activated sludge process regarding sedimentation behaviour.

Contribution of conventional axial computed tomography (nonhelical), in conjunction with panoramic tomography (zonography), in evaluating mandibular fractures.

Previous studies comparing the sensitivity between different radiological exams have concluded that conventional axial computed tomography (CT; nonhelical) is unsuitable in the assessment of mandibular fractures. Axial CT was shown to have a reduced sensitivity compared with plain radiographs and panoramic tomography because it missed nondisplaced fractures in the posterior portion of the mandible. Because the resolution of CT has improved from the time of these previous studies, the authors were interested in assessing whether axial CT (nonhelical) could now provide additional clinically useful information and enhance our understanding of mandibular fractures, beyond that obtained from panoramic tomography alone. In their study, 5 staff surgeons initially evaluated the panoramic tomograms and then the CT scans of 39 patients with 66 fractures. A series of four questions assessed the relative contribution of these two radiological exams in formulating an optimal operative plan for each patient. The authors found that axial CT provided supplementary information regarding missed fractures, comminution, and the exact size and degree of displacement of fracture fragments. This additional data could have changed the operative plan in a substantial proportion of patients (17 of 39). Axial CT demonstrated two missed parasymphyseal fractures (2 of 39 patients) that were not seen on these patients' panoramic tomograms. Axial CT also revealed undiscovered comminution or demonstrated fracture displacement more precisely in 39% of patients (15 of 39) and 24% of fractures (16 of 66). This study demonstrates that axial CT was clinically useful as an additional investigation to panoramic tomography. Axial CT helped elucidate further the nature of suspected mandibular fractures.

Crystallization and preliminary X-ray analysis of the EVH1 domain of Vesl-2b.

Proteins of the Homer/Vesl family are enriched at excitatory synapses and selectively bind to a proline-rich consensus sequence in group 1 metabotropic glutamate receptors via a domain that shows a strong similarity to the Ena/VASP homology 1 (EVH1) domains. EVH1 domains play an important role in actin cytoskeleton dynamics. Crystals of the EVH1 domain of murine Vesl-2b were obtained that diffract X-rays to 2.4 A resolution. They belong to space group C2, with unit-cell parameters a = 112.8, b = 69.9, c = 54.9 A, beta = 110.7 degrees, consistent with three molecules per asymmetric unit and a solvent content of 53%.

Secretion and differential localization of the proteolytic cleavage products Abeta40 and Abeta42 of the Alzheimer amyloid precursor protein in human fetal myogenic cells.

Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease. These peptides are proteolytic cleavage products of the amyloid precursor protein (APP) and are generated by beta- and gamma-secretases. Here we show by multiparameter immunofluorescence imaging in muscle cells that localization of the Abeta40 and Abeta42 cleavage products reveals different myocyte types in a three-dimensional culture system. These myocyte types are heterogeneous by selective intracellular concentration of either Abeta40 or Abeta42 in vesicular structures, whilst only the Abeta40 peptide is secreted as indicated by Western blot analysis. This cellular pattern of APP proteolysis and Abeta peptide secretion correlates with lack of L-APP mRNA splice isoforms. Differential secretion and intracellular accumulation of Abeta peptides is characteristic for the early myocyte development and might be related to cell fusion.

Mitochondrial DNA abnormalities in skeletal muscle of patients with sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a neurodegenerative disease affecting the anterior horn cells of the spinal cord and cortical motor neurons. Previous findings have suggested a specific impairment of mitochondrial function in skeletal muscle of at least a limited number of patients. Applying flavoprotein/NAD(P)H autofluorescence imaging of mitochondrial function in saponin-permeabilized muscle fibres, we detected a heterogeneous distribution of the respiratory chain defect among individual fibres in muscle biopsies of patients (11 out of 17) with sporadic amyotrophic lateral sclerosis (SALS). These findings correlate with the presence of cytochrome c oxidase (COX)-negative muscle fibres detected histologically. We established the molecular basis for the decreased activities of NADH:CoQ oxidoreductase and COX in SALS muscle. In the skeletal muscle of the investigated patients, diminished levels (13 out of 17) or multiple deletions (one out of 17) of mitochondrial DNA (mtDNA) were observed. These alterations of mtDNA seem to be related to decreased levels of membrane-associated mitochondrial Mn-superoxide dismutase. Our results support the viewpoint that an oxygen radical-induced impairment of mtDNA is of pathophysiological significance in the aetiology of at least a subgroup of patients with SALS.