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Demineralized bone matrix (DBM) has been widely investigated as a biomaterial to promote new bone formation and is utilized clinically for bone repair and regeneration. We investigated gene expression patterns of osteogenic differentiation in human periosteal (HPO) cells cultured with demineralized bone matrix, using cDNA array technology. Osteogenic differentiation of HPO cells was determined using alkaline phosphatase assay. In order to examine differential gene expression during osteogenic differentiation, total RNA was isolated from HPO cells in the absence or presence of DBM on day seven and analyzed using osteogenesis cDNA gene array. The selected genes were verified using reverse transcriptase (RT)-PCR analysis. Human periosteal cells differentiated along an osteogenic lineage after treatment of DBM. The alkaline phosphatase activity assay showed that HPO cells differentiated into an osteogenic lineage. Gene expression of HPO cells treated with DBM for seven days was analyzed with cDNA array and RT-PCR analyses. Expression of biglycan, TGF-β1, and TGF-βR1 was upregulated, whereas collagen14A1 expression was downregulated, as confirmed by RT-PCR. Human periosteal cells expressed osteogenesis genes when treated with DBM. These findings provide new insight into the capability of demineralized bone matrix to modulate the osteogenic differentiation of human periosteal cells.

Cryobiology deals primarily with the art of freezing aqueous solutions. These solutions may reside inside or outside of living cells and the transition of this solution from the liquid phase into the solid phase constitutes freezing For purposes of this presentation, the cryobiology of aqueous solutions has been divided into sections with the understanding that they are not distinct from each other In addition, emphasis is placed on selected cells/tissues and on solution of problems encountered in freezing, storage, and thawing of these selected cells/tissues, rather than attempt to provide an all inclusive review of the subject of cryobiology. Hence, the title “Engineering Aspects of Cryobiology” is intended to illustrate how problems encountered in the freezing (cryopreservation) of cells and tissues have been resolved through the engineering of devices used in the various steps of their cryopreservation. This presentation will deal with freezing (with focus on containers, controlled-rate freezers, and temperature monitoring), storage (with focus on mechanical versus liquid nitrogen, thermal/mechanical perturbations, and shipping), and thawing (with focus on rates of thawing, maintenance of sterility, and dilutional removal of cryoprotectants).

Varus tension testing was performed on 26 matched pairs of tibias after high tibial osteotomy with three different fixation techniques--Coventry stepped staple, Mansat staple blade, and modified tension-band. Biomechanical testing revealed that in group I the Coventry stepped staple and Mansat staple blade yielded similar force at failure values of 132.44 +/- 29.29 and 137.34 +/- 40.84, respectively. In group II, the varus force at failure value was 170.45 +/- 83.95 for the modified tension band device versus 115.27 +/- 67.21 for the Coventry stepped staple device. In group III, the varus force at failure value was 180.26 +/- 82.36 for the modified tension band device versus 109.14 +/- 60.96 for the Mansat staple blade. The findings in this study suggest that the modified tension band technique provides a greater varus force at failure value, approximately 160-170%, compared to the other two techniques. In addition, this device is easy to apply and less expensive, and most orthopaedic surgeons are already familiar with its use.

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.

The ability of auxotrophs of Neurospora crassa to grow on certain tripeptides, despite the presence of excess competing amino acids, suggests it has an oligopeptide transport system. In general, dipeptides did not support growth except in those instances where extracellular hydrolysis occurred, or where the dipeptide appeared to be accumulated by an uptake system which is sensitive to inhibition by free amino acids. Considerable intracellular peptidase activity toward a large number of peptides was demonstrated, including a number of peptides which could not be utilized for growth. The intracellular peptidase activity was shown to be selective for amino acid composition and sequence (N-terminal or C-terminal) within the peptide; glycine-containing peptides were particularly poor substrates for peptidase activity. Only a small amount of extracellular peptidase activity could be detected.

Transport of the acidic amino acids by Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The neutral transport system is shown to possess “high affinity”, but low transport capacity for the acidic amino acids. The general transport system is shown to have “low affinity” and high transport capacity for these amino acids. The non-linearity of a Lineweaver-Burk plot of aspartate transport by the wild type has been shown to result from an overlapping of two transport systems for accumulation of this amino acid. Removal, by mutation, of the neutral transport system restores the transport kinetics to a linear function. The combined, and separate, transport activities are shown to be stereospecific, preferring the l-stereoisomeric form over the d-form, and to transport the neutrally charged species of l-aspartate and l-glutamate. The transport activity is dependent on pH, and the V, but not the C12, constant of the general transport system is pH dependent. Two models for amino acid transport are presented as possible mechanisms to explain the observed kinetics. One model involves three genetically distinct transport systems. The second model utilizes a single “allosteric permease” to account for the kinetic data.

1. 1. l -Aspartate transport activity was optimally induced following 6 h growth on acetate minimal media. 2. 2. The observed aspartate (and glutamate) transport activity was attributed to the general amino acid transport system since l -phenylalanine, but not l -cysteic acid, inhibited transport activity at all pH values tested. 3. 3. Changes in K m values for l -aspartate and l -glutamate transport following growth on acetate minimal media indicated that apparent “affinities” of the “acidic amino acid transport systems” dramatically increased during the stages of active growth.

The general amino acid transport system in Neurospora crassa conidia is shown to be stimulated to a greater rate of transport activity by the basic amino acids L-arginine and L-lysine. The neutral amino acid L-phenylalanine causes a reduction in the rate of transport activity by this system. The possible mechanisms of regulation by these amino acids are discussed.

Peptides ranging in size from a mean number of 30 residues down to dipeptides supported growth of a leucine auxotroph when used as both a nitrogen and leucine source. Under nitrogen-limiting conditions, the peptides induced extracellular peptidohydrolytic activity, hydrolyzing peptides to monomer amino acids. Growth of a leu-2 mutant of Neurospora crassa on those peptides transportable by the oligopeptide transport system did not result in induction of hydrolytic activity, whereas growth of a leu-2; gltR mutant on these same peptides resulted in induction of peptidohydrolytic activity. The induced extracellular proteolytic activity was shown to be analogous to that inducible by growth on proteins, e.g., bovine serum albumin.

The filtration enrichment procedure of Catcheside (1954) was employed in the isolation of the tyr-s mutation. The auxotroph, leu-3, was mutagenized with ultraviolet light to greater than go % kill and introduced into a bubble culture containing Vogel's minimal medium N (Vogel, 1956) containing 2 % (w/v) sucrose and the tripeptide glycyl-L-leucyl-L-tyrosine at 0.33 mM. After 5 days of filtration and incubation, the remaining cells were sedimented by centrifugation and spread on sorbose-sucrose (I '5 % : 0.1 %, w/v) minimal medium plates containing L-leucine at 0.33 mM and incubated at 30 O C until colonies appeared. Individual colonies were cultured and retested for growth on the tripeptide and on other peptides containing leucine. All such isolates hydrolysed peptides at a rate comparable to the parent leu-3 and a wild type (suqf,a). The method of testing for growth on various compounds containing leucine or tyrosine has been previously described (Wolfinbarger & Marzluf, 1974). Analysis of amino acid pools was performed on hot water extracts of mycelia using a Beckman 721 amino acid analyser and standard analyser procedures.

Growth of an amino acid auxotroph of Neurospora crassa on oligopeptides is shown to occur by extracellular hydrolysis, with subsequent utilization of monomer amino acid residues, and by transport of peptides. Peptides with a hydrodynamic volume greater than that of trileucine are not transported, and this lack of transport is shown to be due to restriction by the oligopeptide transport system rather than the cell wall.

The tripeptide, glycyl- d , l -leucyl- l -tyrosine was chemically synthesized in radioactive form and used to directly study the specificity, regulation, and properties of an oligopeptide transport system in Neurospora . Transport activity is sensitive to azide but does not result in the accumulation of the intact peptide; rather, the radioactive label is accumulated as free tyrosine. Inhibition studies suggest that the transport system probably has a relatively wide range of specificity and is responsible for uptake of short oligopeptides of quite distinct sequences. However, free amino acids and dipeptides are not transported significantly, if at all, by the oligopeptide transport system. A free amino group appears to be a requirement for peptide transport. A mutant strain that is unable to use various peptides for growth is further described and shown to be reduced greater than 90% in transport of the tripeptide.

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.

Characterization of a double mutant, his-6: hgu-4, which is unable to utilize L-histidyl-glycine as a source of histidine has revealed a new locus on linkage group V. The hgu-4 genotype results in a generalized reduced transport activity for amino acids, with a concomitant increased resistance to amino acid analogs. Transport rates and analog resistance for amino acids by this mutant are compared to the previously reported transport deficient mutants fpr-1, nap and un-3. Transport of L-aspartate as a function of temperature is examined in a variety of transport deficient strains in an attempt to explain the mode of action of mutation which pleiotropically affect several genetically and biochemically distinct amino acid transport systems.

1. 1. The transport of tricarboxylic acid cycle dicarboxylic acids by Neurospora crassa was induced 80-fold by growth on acetate whereas tricarboxylic acid enzymes were induced only 2-6-fold and glyoxylate cycle enzymes were induced 20-fold. 2. 2. From the kinetics of uptake and competitive inhibition it was concluded that a single system was responsible for the transport of succinate, fumarate, l-malate and possibly α-ketoglutarate as well as a number of structural analogues. The system appears to be less specific than the known bacterial systems. 3. 3. Succinate transport was strongly inhibited by various known inhibitors of energy metabolism — primarily by proton conductors. Transport was also severely inhibited by various sulfhydryl reagents. 4. 4. Succinate, fumarate and l-malate uptake were identically pH dependent and evidence is presented which implicates the monoionic species as the molecular form of the dicarboxylic acid transported.

A double amino acid transport-deficient mutant (Pm − NB) of Neurospora crassa is shown to be altered in the molecular structure of its cell wall or membrane. This alteration was revealed by a high degree of cellular disruption and cell-cell interaction following extraction by a high molar concentration of KCl.

Transport of l -aspartic acid in Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The transport activity is dependent on pH and results in accumulation of l -aspartic acid against a gradient. Mutants deficient in transport of l -aspartic acid are described.