Magnesium alloys as implant materials--principles of property design for Mg-RE alloys.

Magnesium alloys have attracted increasing interest in the past years due to their potential as implant materials. This interest is based on the fact that magnesium and its alloys are degradable during their time of service in the human body. Moreover magnesium alloys offer a property profile that is very close or even similar to that of human bone. The chemical composition triggers the resulting microstructure and features of degradation. In addition, the entire manufacturing route has an influence on the morphology of the microstructure after processing. Therefore the composition and the manufacturing route have to be chosen carefully with regard to the requirements of an application. This paper discusses the influence of composition and heat treatments on the microstructure, mechanical properties and corrosion behaviour of cast Mg-Gd alloys. Recommendations are given for the design of future degradable magnesium based implant materials.

Support vector machines for spam categorization.

We study the use of support vector machines (SVM's) in classifying e-mail as spam or nonspam by comparing it to three other classification algorithms: Ripper, Rocchio, and boosting decision trees. These four algorithms were tested on two different data sets: one data set where the number of features were constrained to the 1000 best features and another data set where the dimensionality was over 7000. SVM's performed best when using binary features. For both data sets, boosting trees and SVM's had acceptable test performance in terms of accuracy and speed. However, SVM's had significantly less training time.

Improving generalization performance using double backpropagation.

In order to generalize from a training set to a test set, it is desirable that small changes in the input space of a pattern do not change the output components. This can be done by forcing this behavior as part of the training algorithm. This is done in double backpropagation by forming an energy function that is the sum of the normal energy term found in backpropagation and an additional term that is a function of the Jacobian. Significant improvement is shown with different architectures and different test sets, especially with architectures that had previously been shown to have very good performance when trained using backpropagation. It is shown that double backpropagation, as compared to backpropagation, creates weights that are smaller, thereby causing the output of the neurons to spend more time in the linear region.

Characteristics of chloride-dependent incorporation of glutamate into brain membranes argue against a receptor binding site.

Although membrane sites from brain, labelled with [3H]glutamate (Glu) under sodium-free conditions, are thought to represent excitatory receptors, certain anomalous characteristics of the kinetics of apparent binding raised the question of whether transport might contribute to this process, prompting a closer examination of it. Hyperosmolar media and low incubation temperatures (4 degrees C) both led to decreases in the apparent specific binding of [3H]glutamate to membranes from the brain of the rat in the presence of chloride. Furthermore, only 15% of the [3H]glutamate, bound at 37 degrees C, was dissociable when the membranes were then cooled to 4 degrees C. The binding of [3H]glutamate was increased in the presence of certain dipeptides such as L-phenylalanyl-L-glutamate (Phe-Glu); and the binding augmented by the presence of Phe-Glu, was also sensitive to temperature and osmolarity of the incubation buffer. Sonication of membranes in 5 mM glutamate increased the apparent binding of [3H]glutamate and abolished the stimulatory effect of Phe-Glu. These findings are consistent with the hypothesis that chloride-dependent association of [3H]glutamate with membranes from brain reflects, in part, a sequestration process, which may be driven by glutamate exchange.

Quisqualate-sensitive, chloride-dependent transport of glutamate into rat brain synaptosomes.

A chloride-dependent transport process for glutamate has been identified in partially purified rat brain synaptosomes. This process shares many characteristics with the chloride-dependent sequestration process for glutamate in brain sonicates, which was previously thought to represent a quisqualate receptor, such as sensitivity to specific inhibitors and regulation by anions. Increasing the concentrations of chloride led to an increase in the apparent Vmax without affecting the KT. Synaptosomes preincubated with [3H]-L-glutamate exhibit an efflux of the radiolabel, which was stimulated by a substrate for the carrier in the incubating medium, indicating the bidirectional nature of the transport. The chloride-dependent transfer process is restricted to the brain, and regional and developmental profiles clearly distinguish it from the sodium-dependent high-affinity uptake process for glutamate. Nevertheless, the effects of excitotoxic lesions strongly suggest a neuronal localization of the chloride-dependent transport.

Characterization and comparison of a Neurospora crassa RNase purified from cultures undergoing each of three different states of derepression.

Extracellular RNase N4 from Neurospora crassa is derepressible by limitation of any of the three nutrient elements obtainable from RNA. We have purified and characterized the enzyme from cultures grown under each of the three states of derepression. The purification procedure consisted of an ultrafiltration step, cation-exchange chromatography, and gel filtration. We found only one enzyme (N4) that hydrolyzed RNA at pH 7.5 in the presence of EDTA in culture filtrates from nitrogen-, phosphorus-, or carbon-limited cells. In all three cases, the enzymes were identical by polyacrylamide gel electrophoresis (Mr approximately 9,500) and by gel filtration (Mr approximately 10,000). There were no differences in thermal stability or pH optimum; all three cross-reacted with antibody to the nitrogen-depressed enzyme in interfacial ring and in Ouchterlony tests. Digestion of homopolyribonucleotides indicated that N4 preferentially cleaved phosphodiester bonds adjacent to guanine residues. Results indicate that the enzymes are very similar or identical and are probably products of the same gene. N4 appears to be homologous to guanine-specific RNases from other fungal sources.

Regulation of a Neurospora crassa extracellular RNase by phosphorus, nitrogen, and carbon derepressions.

A new extracellular RNase, designated N4, was detected in culture filtrates from Neurospora crassa and its regulation was studied. Limitation of a nutrient obtainable from RNA alone was not sufficient to cause enzyme derepression. The addition of RNA to the medium had no inductive effect, but the addition of exogenous protein caused enzyme production. With protein in the medium, N4 was derepressible for all three elemental nutrients obtainable from RNA: carbon, nitrogen, and phosphorus. Successful carbon derepression required the addition of a small amount of proteolytic activity to the cultures, as has been reported for the carbon-derepressible proteases of N. crassa. Exogenous protein affected RNase production before translation. Effects of the exogenous protein appeared similar to those previously reported for N. crassa protease induction. N4 was under the control of the nit-2 and nuc-1 gene products. nit-2 and nuc-1 mutants were unable to derepress enzyme synthesis for nitrogen and phosphorus limitation, respectively; however, these mutants responded like wild types to the other two states of derepression. Enzyme synthesis was constitutive in the preg mutant. Results indicate that the transcription of the N4 structural gene responds to multiple regulatory gene products from different regulatory circuits and that external protein affects the synthesis of classes of hydrolases other than proteases.

Purification and characterization of an extracellular acid protease from Neurospora crassa.

An extracellular acid protease was purified 1420-fold from sulfur-starved protein-induced cultures of Neurospora crassa. The enzyme was homogeneous as determined by polyacrylamide electrophoresis. The purification procedure consisted of an ultrafiltration step, cation-exchange chromatography, and affinity chromatography on Sepharose-linked pepstatin. The enzyme is homologous to aspartyl proteases that are characterized by pepstatin inhibition and trypsinogen activation. It is extremely autolytic, especially under denaturing conditions. The protease is stable between pH 3 and 7, showing optimal activity near pH 4.0 for both trypsinogen activation and hydrolysis of bovine serum albumin. The molecular weight of the enzyme was 34,500 by gel electrophoresis and gel filtration, and 34,975 by amino acid analysis.

Extracellular acid proteases from Neurospora crassa.

Three electrophoretically distinct acid proteases appear in culture filtrates of Neurospora crassa. Like the previously investigated alkaline and neutral proteases, these enzymes require induction by an exogenous protein. But in contrast to alkaline and neutral proteases, which are synthesized and secreted in response to limitation of any one of three nutrilites (carbon, nitrogen or sulfur), extracellular elaboration of the acidic proteases is more specifically a function of the missing nutrilite. AcP, a pepstatin-inhibitable enzyme similar to other fungal carboxyl proteases, was secreted in large amounts when protein was the sole source of sulfur. Only trace amounts were secreted when nitrogen was the limiting nutrilite, and it was undetectable under carbon limitation. M-1, a chelator-sensitive protease, was secreted when nitrogen or carbon was limiting. M-2, also chelator sensitive, was present only when nitrogen or sulfur was limiting. The evidence presented suggests that the differential regulation of the acidic proteases with respect to nutrilite deprivation may not occur at the level of transcription. AcP and M-2 were partially purified from nitrogen-derepressed cultures by ultrafiltration, cation-exchange chromatography, and gel filtration. AcP has a molecular weight of 66,000, is stable from pH 3.0 to 6.0, and is optimally active toward bovine serum albumin at pH 4.0. M-2 has a molecular weight of 18,000, is stable from pH 1.6 to 5.5, and has optimal activity at pH 4.5.

Alkaline protease from Neurospora crassa. Purification and partial characterization.

A simple purification procedure has been developed for the extracellular alkaline protease from Neurospora crassa. Key steps in the purification were: 1) the choice of gelatin as the protein inducer, which induces optimally at a much lower concentration than other commonly employed protein inducers; 2) heat treatment, during which the inducer is digested by the protease; and 3) a concentration step that eliminates the usual precipitation procedures and removes much of the digested protein inducer. These procedures were followed by routine ion exchange chromatography and gel filtration. The preparation was homogeneous, as determined by gel electrophoresis and ultracentrifugal analyses. A molecular weight of approximately 30,500 was determined by amino acid analysis, gel electrophoresis, and sedimentation equilibrium. The protease has 100% activity from pH 6.0 to 10.0, is heat labile above 45 degrees C, and susceptible to autodigestion. Hydrolysis of the beta chain from insulin indicates a preferential cleavage on the carboxyl group side of neutral and aromatic amino acids.

Use of thermolysin for the dissociation of lung tissue into cellular components.

A method is described for the dissociation of rat lungs into individual viable cells. Thermolysin was perfused through the vasculature and trachea. The lung was then minced and further dissociated by washing with sequential additions of thermolysin. The results indicate this procedure to be an effective means of dispersing lung tissue into its cellular components. Standard trypsinizing procedures generally yield approximately 5 per cent of the total available lung cells whereas the thermolysin treatment increases cell yield 10-fold without significantly affecting cell viability.

Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process.

To induce exocellular proteolytic enzyme from carbon-starved exponential-phase cells of Neurospora crassa, both a protein substrate and an activating protease of certain specific properties must be present at the same time. The cells must be capable of protein synthesis, since cycloheximide inhibits the process, but cell growth, as determined by increase in cell mass, does not appear to be required. Both soluble (bovine serum albumin, myoglobin) and insoluble protein substrates (collagen, corn zein) will affect protease induction, although certain soluble, globular proteins (egg white globulin, bovine gamma globulin) will not. In most cases, rates of protease induction are proportional to protein concentration, regardless of the nature of the inducing protein. All activating proteases capable of affecting induction in a manner similar to that of N. crassa exocellular protease were of bacterial origin and were exoproteases. Mammalian proteases and peptidases had little or no effect on the induction process.

Regulation of exocellular proteases in Neurospora crassa: role of Neurospora proteases in induction.

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.