Detectivity enhancement in quantum well infrared photodetectors utilizing a photonic crystal slab resonator.

We characterize the performance of a quantum well infrared photodetector (QWIP), which is fabricated as a photonic crystal slab (PCS) resonator. The strongest resonance of the PCS is designed to coincide with the absorption peak frequency at 7.6 µm of the QWIP. To accurately characterize the detector performance, it is illuminated by using single mode mid-infrared lasers. The strong resonant absorption enhancement yields a detectivity increase of up to 20 times. This enhancement is a combined effect of increased responsivity and noise current reduction. With increasing temperature, we observe a red shift of the PCS-QWIP resonance peak of -0.055 cm(-1)/K. We attribute this effect to a refractive index change and present a model based on the revised plane wave method.

Crosslinking kinetics of the human transglutaminase, factor XIII[A2], acting on fibrin gels and gamma-chain peptides.

Factor XIII is the terminal enzyme of the coagulation cascade which serves to rapidly crosslink the adjacent gamma-chain C-termini of fibrin clots. In vivo, this process is initiated by the proteolytic action of thrombin which simultaneously converts both soluble fibrinogen to fibrin and activates zymogen FXIII; fibrin then spontaneously polymerizes to form a gel which activated FXIII stabilizes through crosslinking. Due to the kinetic complexity and the difficulty of investigating gel phase reactions, methods employing pre-activation of recombinant human Factor XIII (rFXIII[A'2]) were developed to effectively decouple these reactions. By utilizing these methods, the kinetic parameters of gamma-chain crosslinking in fibrin gels could be determined by both initial rate and integrated rate techniques under physiologically relevant conditions. The crosslinking of the gamma-chain of fibrin gels could be described by apparent Michaelis kinetics with K(m)(app) = 6.2 microM, kcat = 1872 min-1, and Ksp = 302 min-1 microM-1 for a fibrin gamma-chain monomer of M(r) = 170000 Da. In contrast, both the crosslinking rates of alpha-chains within fibrin gels (Ksp = 0.38 min-1 microM-1: Bishop et al. (1993)) and the crosslinking of a soluble synthetic peptide containing the unique gamma-chain fibrin crosslinking site (Ksp = 0.030 min-1 microM-1) could not be shown to saturate and gave apparent first-order rates with respect to rFXIII[A'2]. These observations coupled with the large differences in the turnover rates (approximately 10(4)) suggest two likely mechanisms for FXIII[A'2]-substrate interactions: (1) random (or independent) binding of non- or weakly interacting substrate pairs imposes a high entropic barrier (i. e., delta Gbinding) to the formation of a productive catalytic complex, e.g., for soluble gamma-chain peptides and the flexible alpha-chains within fibrin, and (2) binding to an oriented substrate pair effectively lowers the entropic barrier to formation of a Michaelis complex and thus greatly enhances the rate of catalysis, e.g., for gamma-chain pairs within the fibrin fibrils.

High-level expression of recombinant human fibrinogen in the milk of transgenic mice.

Fibrinogen is a complex plasma protein composed of two each of three different polypeptide chains. We have targeted expression of r-human fibrinogen to the mammary gland of transgenic mice. Three expression cassettes, each containing the genomic sequence for one of the three human fibrinogen chains controlled by sheep whey protein beta-lactoglobulin promoter sequences, were coinjected into fertile mouse eggs. Southern blot analysis demonstrated that more than 80% of the transgenic founders contained all three fibrinogen genes. Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of milk from the highest producing founder animal demonstrated the presence of human fibrinogen subunits at concentrations of 2000 micrograms/ml. In several animals with a balanced ratio of the individual fibrinogen subunits, up to 100% of the protein was incorporated into fully assembled fibrinogen hexamers. Incubation of the transgenic milk with thrombin and factor XIII resulted in a cross-linked fibrin clot, indicating that a major portion of the secreted fibrinogen was functional. These studies represent the first report of high-level biosynthesis and secretion of a functional, complex, hexameric protein in the milk of a transgenic animal.

Aqueous stability of recombinant human thrombopoietin as a function of processing schemes.

Preformulation studies conducted with recombinant human thrombopoietin (rhTPO), a 332 amino acid glycoprotein which stimulates platelet production, show distinctions in degradation profiles as a function of processing schemes. The stability-limiting degradation pathways change as a function of purification stage and method and are dependent upon the presence of contaminating protease. The stability-limiting degradation pathway of affinity-purified and in-process rhTPO preparations is primarily attributed to proteolysis initiated by a protease present as a fermentation contaminant. The proteolysis increases with increasing pH as a function of temperature. The degradation profiles for these preparations show that bioactivity initially increases and then decreases with increasing pH as a function of temperature. This is consistent with proteolysis to active forms which ultimately undergo degradation to less active forms. Similar studies conducted with rhTPO preparations purified by a combination of more conventional chromatographic steps show different stability-limiting degradation pathways and a different pH-stability profile when compared to affinity purified or in-process preparations. In this case, degradation is accompanied by decreases in activity under all conditions, consistent with the conversion to less active forms. These results illustrate the importance of preformulation and stability characterization of protein pharmaceuticals in support of both process and formulation development. Issues related to storage and handling of inprocess preparations differ from those with formulated product since the stability-limiting degradation pathways change as a function of purification stage.

Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice.

Correction of the obese state induced by genetic leptin deficiency reduces elevated levels of both blood glucose and hypothalamic neuropeptide Y (NPY) mRNA in ob/ob mice. To determine whether these responses are due to a specific action of leptin or to the reversal of the obese state, we investigated the specificity of the effect of systemic leptin administration to ob/ob mice (n = 8) on levels of plasma glucose and insulin and on hypothalamic expression of NPY mRNA. Saline-treated controls were either fed ad libitum (n = 8) or pair-fed to the intake of the leptin-treated group (n = 8) to control for changes of food intake induced by leptin. The specificity of the effect of leptin was further assessed by 1) measuring NPY gene expression in db/db mice (n = 6) that are resistant to leptin, 2) measuring NPY gene expression in brain areas outside the hypothalamus, and 3) measuring the effect of leptin administration on hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Five daily intraperitoneal injections of recombinant mouse leptin (150 micrograms) in ob/ob mice lowered food intake by 56% (P < 0.05), body weight by 4.1% (P < 0.05), and levels of NPY mRNA in the hypothalamic arcuate nucleus by 42.3% (P < 0.05) as compared with saline-treated controls. Pair-feeding of ob/ob mice to the intake of leptin-treated animals produced equivalent weight loss, but did not alter expression of NPY mRNA in the arcuate nucleus. Leptin administration was also without effect on food intake, body weight, or NPY mRNA levels in the arcuate nucleus of db/db mice. In ob/ob mice, leptin did not alter NPY mRNA levels in cerebral cortex or hippocampus or the expression of CRH mRNA in the hypothalamic paraventricular nucleus (PVN). Leptin administration to ob/ob mice also markedly reduced serum glucose (8.3 +/- 1.2 vs. 24.5 +/- 3.8 mmol/l; P < 0.01) and insulin levels (7,263 +/- 1,309 vs. 3,150 +/- 780 pmol/l), but was ineffective in db/db mice. Pair-fed mice experienced reductions of glucose and insulin levels that were < 60% of the reduction induced by leptin. The results suggest that in ob/ob mice, systemic administration of leptin inhibits NPY gene overexpression through a specific action in the arcuate nucleus and exerts a hypoglycemic action that is partly independent of its weight-reducing effects. Furthermore, both effects occur before reversal of the obesity syndrome. Defective leptin signaling due to either leptin deficiency (in ob/ob mice) or leptin resistance (in db/db mice) therefore leads directly to hyperglycemia and the overexpression of hypothalamic NPY that is implicated in the pathogenesis of the obesity syndrome.

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

Human recombinant factor XIII from Saccharomyces cerevisiae. Crystallization and preliminary x-ray data.

Crystals of human recombinant factor XIII from the yeast Saccharomyces cerevisiae have been grown from solutions of ammonium sulfate at pH 5.8. The crystals are orthorhombic, with space group P2(1)2(1)2 and unit cell dimensions gamma a = 101.2, b = 182.7, and c = 93.4 A. The asymmetric unit consists of one a2 dimer of molecular mass 166 kDa. A 3.5-A resolution data set for the native protein has been collected. Practical resolution limits for these crystals have not been determined, but reflections have been observed to a Bragg spacing of 2.8-A resolution.

Expression, purification, and characterization of human factor XIII in Saccharomyces cerevisiae.

Factor XIII is the terminal enzyme of the clotting cascade. A cDNA sequence encoding human placental factor XIII was expressed in Saccharomyces cerevisiae with the yeast ADH2-4c promoter. Expression levels were a strong function of the noncoding flanking DNA content of the construction. When the terminal 3'-flanking noncoding DNA was removed, expression increased approximately 50-fold. The protein was produced in quantity by high-yield fermentation and purified to homogeneity. The recombinant protein was cleaved by thrombin at the same activation site as purified human placental FXIII and exhibited 100% enzymatic activity. At high thrombin concentrations rFXIIIa was cleaved into inactive 54- and 25-kDa polypeptides. The identity of these cleavage sites and the blocked N-terminus to that of the human protein was revealed by amino acid microsequencing. A time course of thrombin activation was performed and the relative distribution of the thrombin-cleaved subunits to the uncleaved zymogen subunits determined; the results were consistent with the half of the sites catalytic model for transglutaminase activity proposed by Chung et al. (Chung, S. I., Lewis, M. S., & Folk, J. E. (1974) J. Biol. Chem. 249, 940-950, 1974) and Hornyak et al. (Hornyak, T. J., Bishop, P. D., & Shafer, J. A. (1989) Biochemistry 28, 7326-7332). Equilibrium and velocity sedimentation analysis indicated that rFXIII exists as a 166-kDa nondissociating dimer that behaves as a compact particle of 8.02 S. Thus, all of the properties of rFXIII thus far examined are consistent with those reported for human platelet and placental FXIII.(ABSTRACT TRUNCATED AT 250 WORDS)

T4 phage deoxyribonucleoside triphosphate synthetase: purification of an enzyme complex and identification of gene products required for integrity.

We have isolated a highly enriched preparation of the multienzyme complex which synthesizes deoxyribonucleoside triphosphates (dNTPs) from bacteriophage T4-infected bacteria. By a combination of SDS polyacrylamide gel electrophoresis and assays for specific enzyme activities, we have been able to identify in our final preparation ten different gene products which were previously identified as constituents of this complex, based upon studies with crude preparations. The complex dissociates at high concentrations of NaCl and MgCl2 but is stable under ionic conditions thought to exist in vivo. The purified complex catalyzes the efficient five-step conversion of dCTP to dTTP. Experiments with several T4 mutants have demonstrated that gene products encoded by cd, regA, nrdA, and nrdB are necessary to retain physical integrity of the complex throughout the preparative procedure, while gp44, gp55, and gppseT are not required. We conclude from this evidence that the T4 early gene products which function in dNTP biosynthesis are, in fact, physically linked as a multienzyme complex, and that regA contributes to the integrity of this complex. However, the dNTP-synthesizing complex as we isolate it contains no detectable DNA polymerase, nor have other known replication proteins been detected.

T4 phage-coded dihydrofolate reductase. Subunit composition and cloning of its structural gene.

The structures of phage-coded dihydrofolate reductases are of interest because of 1) possible relationship to plasmid-coded dihydrofolate reductases; 2) unusual regulation of enzyme synthesis; and 3) multiple roles and intermolecular interactions involving the protein. To prepare for primary structural studies, we have cloned the T4 frd gene, which codes for dihydrofolate reductase, and we have determined for redetermined some physical properties of the enzyme. The native enzyme has a molecular weight of about 44,500, as determined by sedimentation velocity and gel filtration, and a subunit molecular weight of about 23,000, as determined by aminopterin titration and denaturing gel electrophoresis. We conclude that the enzyme is a dimer, with each subunit containing one methotrexate-binding site. A 1.1-kilobase pair fragment from a HindIII restriction digest of cytosine-substituted T4 DNA was cloned into pBR322, and recombinants were identified by trimethoprim resistance. Cells carrying this recombinant plasmid produce both the host cell and phage-coded dihydrofolate reductases.

T4 ribonucleotide reductase. Physical and kinetic linkage to other enzymes of deoxyribonucleotide biosynthesis.

This laboratory has described a multienzyme aggregate from T4 phage-infected Escherichia coli which seems to participate in deoxyribonucleotide biosynthesis and efficient delivery of DNA precursors to the replication apparatus. This paper describes improved methodology for isolation of this aggregate, and we present three lines of evidence supporting a role for ribonucleoside diphosphate reductase in functioning of the presumed complex. 1) Ribonucleoside diphosphates are readily incorporated into DNA as deoxyribonucleotides in an in situ DNA-synthesizing system from T4 phage-infected cells. 2)Ribonucleotide reductase is associated with the complex, as shown by co-sedimentation of reductase activity with other activities in the multienzyme aggregate we have described. 3)Ribonucleotide reductase is kinetically coupled to at least four other enzymes involved in a sequential pathway. The aggregated enzymes catalyze the five-step conversion of uridine diphosphate to deoxythymidine triphosphate with but a brief lag before dTTP production reaches its maximal rate. These studies have also confirmed the existence of dCTPase-dUTPase and dCMP deaminase activities in the putative complex.