Microwave absorption of gamma'-Fe2.6 Ni1.4N nanoparticles derived from nitriding counterpart precursor.

Gamma-Fe2.6Ni1.4 nanoparticles were prepared by the arc-discharge method as the precursor and its nitride counterpart of gamma'-Fe2.6Ni14N nanoparticles was synthesized directly through a thermal ammonolysis reaction at the temperature of 673 K for two hours. The resultant product was identified as a homogeneous ternary nitride with nearly spherical shape and average size of about 60.0 nm. The electromagnetic characteristics of gamma'-Fe2.6Ni1.4N derivant and gamma-Fe2.6Ni1.4 precursor have been studied in the frequency range of 2-18 GHz. Compared with the precursor, gamma'-Fe2.6Ni1.4N nanoparticles exhibits an enhanced electromagnetic absorption property resulted from the increased dielectric loss by nitriding process. The optimal reflection loss (RL) of gamma'-Fe2.6Ni1.4N nanoparticles/paraffin composite can reach -39.9 dB at 5.2 GHz in a thickness of 2.29 mm, and the frequency band corresponding RL < -10 dB is over 2.6-18 GHz in the thickness range of 0.78-4.20 mm.

Optimization of microwave pretreatment conditions to maximize methane production and methane yield in mesophilic anaerobic sludge digestion.

The objective of this study was to find optimum microwave pretreatment conditions for methane production and methane yield in anaerobic sludge digestion. The sludge was pretreated using a laboratory-scale industrial microwave unit (2450 MHz frequency). Microwave temperature increase rate (TIR) (2.9-17.1 degrees C/min) and final temperature (FT) (52-108 degrees C) significantly affected solubilization, methane production, and methane yield. Solubilization degree (soluble chemical oxygen demand (COD)/total COD) in the pretreated sludge (3.3-14.7%) was clearly higher than that in the raw sludge (2.6%). Within the design boundaries, the optimum conditions for maximum methane production (2.02 L/L) were TIR = 9.1 degrees C/min and FT = 90 degrees C, and the optimum conditions for maximum methane yield (809 mL/g VS(removed)) were TIR 7.1 degrees C/min and FT = 92 degrees C.

Auxin biosynthesis in maize.

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.

The Drosophila tissue polarity gene inturned functions prior to wing hair morphogenesis in the regulation of hair polarity and number.

The adult cuticular wing of Drosophila is covered with an array of distally pointing hairs. Mutations in the inturned (in) gene result in both abnormal hair polarity (i.e., hairs no longer point distally), and, in most cells forming more than one hair. We have isolated and characterized a collection of in alleles. Among this collection of alleles are a number of rearrangements that enable us to assign in to 77B3-5. Almost all of the in alleles, including putative null alleles, result in a stronger phenotype on the wing at 18 degrees than 29 degrees. The data argue that the in-dependent process is cold-sensitive. Temperature shift experiments with a hypomorphic allele show that this cold sensitivity can be relieved by several hours of incubation at the permissive temperature at a variety of times in the early pupae, but that this ability ends prior to the start of hair morphogenesis. One new allele showed a dramatic heat sensitivity. Temperature shift experiments with this allele revealed a very short temperature-sensitive period that is a few hours prior to the start of hair morphogenesis. That the temperature during hair morphogenesis is irrelevant for the phenotype of in is consistent with the hypothesis that the only role that in has in wing hair development is to regulate the initiation of hair morphogenesis.

Molecular structure of frizzled, a Drosophila tissue polarity gene.

The function of the frizzled (fz) locus is required to coordinate the cytoskeletons of pupal epidermal cells so that a parallel array of cuticular hairs and bristles is produced. We report here the molecular cloning and characterization of the fz locus. The locus is very large. Mutations that inactivate the gene are spread over 100 kb of genomic DNA. The major mRNA product of the gene is a 4-kb RNA that is encoded by 5 exons spread over more than 90 kb of genomic DNA. Conceptual translation of this mRNA indicates that it encodes an integral membrane protein that is likely to contain both extracellular and cytoplasmic domains.

The frizzled gene of Drosophila encodes a membrane protein with an odd number of transmembrane domains.

The protein encoded by the Drosophila tissue polarity gene, frizzled (fz), is required for both the intercellular transmission and the intracellular transduction of a tissue polarity signal. In order to study the biochemical characteristics of this rare protein, we constructed a hs-fz fusion gene and transferred this to Drosophila tissue culture cells and embryos. Cell fractionation experiments and immunostaining experiments showed that the Fz protein is an integral membrane protein containing an odd number of transmembrane domains, consistent with previous suggestions that it contains seven transmembrane domains. Immunostaining of pupal wings showed that the Fz protein is evenly distributed throughout the wing arguing that the Fz protein does not act as a graded morphogen.

Cloning of the genes encoding mouse cardiac and skeletal calsequestrins: expression pattern during embryogenesis.

Calsequestrin is a low-affinity and high-capacity calcium-binding protein in the sarcoplasmic reticulum (SR). In the present study, we have cloned and sequenced mouse cardiac and skeletal calsequestrin cDNAs. The deduced amino acid sequences are highly homologous to those of other mammalian calsequestrins. As expected, the cardiac and skeletal calsequestrins are expressed specifically and exclusively in adult heart and skeletal muscles, respectively. In-situ hybridization was performed to examine the expression pattern of the calsequestrins in the developing mouse and rat embryos. During early organogenesis, the cardiac and skeletal calsequestrin transcripts were detected exclusively in the heart primordium and the myotome of somites, respectively. The cardiac calsequestrin transcripts were later detected in fetal heart and skeletal muscles, whereas the skeletal calsequestrin transcripts were only found in fetal skeletal muscles. These data suggest that the cardiac calsequestrin plays a role in the differentiation and function of heart, and in the function of fetal skeletal muscles in conjunction with the skeletal calsequestrin, but not in the early differentiation of the myotome of somites. The expression of the skeletal calsequestrin in the myotome is regulated probably by myogenin, a myogenic regulatory gene.

New E. coli cloning vector using a cellulase gene (celA) as a screening marker.

The extracellular endoglucanase A gene of Clostridium thermocellum (celA) was used as a screening marker for E. coli cloning vector A 1.4-kb EcoRI fragment containing celA from pTvec/celA was isolated and cloned into a pUC18 deleting beta-galactosidase gene fragment. The constructed vectors, pCEL1, pCEL10, pCEL11, and pCEL20, have different multiple cloning sites within celA. If the cellulase, CelA, is inactivated by insertion of a foreign DNA fragment into multiple cloning sites, the recombinant transformants show no clear halos on an agar plate containing cellulose. This process overcomes the ambiguity of color screening in the X-gal/beta-galactosidase system, and over 90% of the recombinant transformants with no halos have foreign DNA inserts. Several E. coli strains were transformed successfully with pCEL series vectors regardless of mutation for alpha-complementation. Because E. coli strains do not have a cellulase gene, a vector using a cellulase gene screening marker can be used in any E. coli strain without limit. The new cloning system is very efficient, convenient, and cost effective.

Frizzled-suc2 fusion gene studies in Saccharomyces cerevisiae.

The frizzled (fz) gene is required for development of planar tissue polarity in the epidermis of Drosophila melanogaster; it likely encodes an integral membrane protein that functions as a receptor for a tissue polarity-signaling molecule. On the basis of hydropathy analyses, it has been postulated that Fz protein contains seven transmembrane domains. Consistent with that model, the amino terminus of Fz was found to be extracellular, whereas the carboxyl terminus was intracellular. In the present study, the membrane topology of Fz was further examined in vivo by constructing various fz-suc2 fusion genes and analyzing their invertase activity in transformed yeast. We observed that plating efficiency and growth rate on sucrose media, as well as invertase secretion, were consistent with the proposed seven-pass model of Fz. This study provides the first experimental assessment of overall Fz topology.

An improved strategy for a genetic assay for site-specific proteolysis.

We have previously reported a genetic assay that is suitable for the study of substrate specificity of a protease in vivo, and herein present a simplified version of the method. In this procedure, expressed in Saccharomyces cerevisiae by using the constitutive alcohol dehydrogenase promoter is a fusion protein in which a transcription factor is linked to the intracellular domain of an integral membrane protein by a protease substrate sequence. Following this, a protease is expressed by using the inducible GAL promoter in the same yeast cells. The cleavage of the substrate sequence by the specific protease results in the release of the transcription factor and subsequent activation of reporter genes in nucleus. Since the expression of a protease is strictly under the control of the inducible GAL promoter, false substrate sequences that are cleaved by endogenous yeast proteases can be easily recognized and eliminated from further characterization. This suggests that the modified strategy provides an efficient tool for the analysis of substrate sequences of a protease in vivo.

Expression of excitation-contraction coupling proteins during muscle differentiation.

The goal of this study was to characterize three major excitation-contraction (E-C) coupling proteins: ryanodine receptor [RyR, the calcium release channel in the sarcoplasmic reticulum (SR)], dihydropyridine receptor (DHPR, the voltage-gated L-type calcium channel in the transverse tubule) and SR Ca2+-ATPase (SERCA, the calcium pump in the SR) in the differentiating primary cultures of rat skeletal and cardiac muscle cells. The expression levels of these proteins were determined by ligand binding assays and/ or immunoblottings along with an examination of the morphological changes in differentiating muscles by phase-contrast microscopy. In the skeletal cells, the expression levels of the E-C coupling proteins generally increased in the course of differentiation with the peak expression at the 9th day of culture. Simultaneous with the increased expression of the proteins, the myoblasts elongated, followed by alignment and fusion of the cells to form multinucleated myotubes. In the cardiac cells, on the contrary, the peak expression levels of DHPR, SERCA and RyR were reached within 2, 4, and 7 d of culture, respectively. Alignment of the cardiac muscle cells and spontaneous contraction occurred as early as several h after plating. These results suggest that expression patterns of E-C coupling proteins are different between skeletal and cardiac muscles, and that DHPR could play an important role in Ca2+ metabolism during the early stages of differentiation.

Selection of Drosophila genes encoding secreted and membrane proteins.

With the use of a yeast-based signal sequence trap (YSST) method, we screened a Drosophila cDNA library to isolate genes encoding secreted and membrane proteins. Of the 136 unique cDNA clones sequenced, 11 clones (8.1%) are identical to previously known Drosophila genes, 18 clones (13.2%) are homologous to other genes identified in various organisms, and 91 clones (66.9%) are novel. Most of these genes are secreted or membrane proteins, or appear to contain putative signal sequences at their amino termini. This indicates that YSST is an effective tool for the isolation and analysis of Drosophila genes that play roles in intercellular communication.

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans.

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.

Human anti-Pseudomonas aeruginosa outer membrane proteins IgG cross-protective against infection with heterologous immunotype strains of P. aeruginosa.

In order to develop an effective means to treat and prevent Pseudomonas aeruginosa infections, we have purified P. aeruginosa outer membrane protein (Oprs)-specific human IgG antibody using a large-scale affinity column. In this study, we investigated the cross-protective activity of the purified anti-Oprs IgG against various immunotype strains of P. aeruginosa. The anti-Oprs IgG reacted with Oprs isolated from seven Fisher-Devlin immunotype strains of P. aeruginosa and was able to promote opsonophagocytic killing of all seven immunotype strains by human phagocytic cells. Administration of 500 microg anti-Oprs IgG to mice raised the LD50 of the P. aeruginosa strains by 8-250-fold, indicating the protective capacity against heterologous P. aeruginosa strains as well as homologous strains. In contrast, despite high titers against P. (aeruginosa Oprs, total serum IgG isolated from burn patient sera was no better than normal serum IgG in protecting mice from infection with P. aeruginosa. These data demonstrate that the affinity-purified human anti-Oprs IgG could afford protection against heterologous immunotype P. aeruginosa strains and provide a rationale to use anti-Oprs IgG as an adjunct for treatment of P. aeruginosa infections in humans.

Anchor epicanthoplasty combined with out-fold type double eyelidplasty for Asians: do we have to make an additional scar to correct the Asian epicanthal fold?

The epicanthal fold along with a lack of a superior palpebral fold, excessive fat, and laxity of pretarsal skin represent the ethnic characteristics and a traditional sense of beauty in the Asian upper eyelid. But, too prominent an epicanthal fold may ruin an otherwise beautiful eye; furthermore, it becomes a restriction that makes the out-fold type double eyelidplasty, one of the two main types of double eyelidplasty, impossible. If a double eyelid as an out-fold type is desired, a concomitant epicanthoplasty should be performed with the possibility of hypertrophic scarring of the medial canthal area in Asians. To address the Asian epicanthal fold without danger of hypertrophic scarring, the authors developed an anchor epicanthoplasty technique that leaves no additional scar when combined with a double eyelidplasty. This technique is based on the concept of trimming of muscle and soft tissue under the Asian epicanthal fold and downward medial advancement and anchoring of the medial canthal skin to the deep tissue. The technique consists of five procedures based on the assumed causes of the Asian epicanthal fold: (1) augmentation rhinoplasty, (2) downward medial advancement of the medial upper lid skin, (3) removal of the superficial insertion of the medial canthal ligament and selective removal of the orbicularis oculi muscle, (4) subcutaneous contouring of the thick nasal skin, and (5) anchoring of the medial end of the incision to the deep tissue. During the past 12 years (1988 to 1999), 67 anchor epicanthoplasty procedures have been performed. Twenty-eight cases were followed up for more than 3 months, and all of the patients were satisfied with the results. There were only a few minor complications, which could be corrected with minimal revision. As an ancillary procedure to a double eyelidplasty, this anchor epicanthoplasty can reduce the Asian epicanthal fold and make a double fold as an out-fold type without an additional scar. In terms of hypertrophic scarring and compatibility with out-fold type double eyelidplasty, this anchor epicanthoplasty is the best method for correcting Asian epicanthal fold compared with other preexisting procedures. Other advantages of this technique are a wide range of applications and no compromise of medial, canthal skin to interfere with other epicanthoplasty techniques. Some disadvantages of this technique are technical difficulty and the possibility of active bleeding.

Simplified anatomic method of double-eyelid operation: septodermal fixation technique.

Conventionally, it has been argued that the aponeurosis meets the orbital septum below the upper tarsal border in Orientals. However, we have shown that the aponeurosis fused with the orbital septum above the upper tarsal border in 502 patients, which contradicts the classic concept. In 457 patients there were distinct layers of fascia anterior to the orbital septum that originate from the septum and insert onto the pretarsal aponeurotic expansion. In Oriental eyelids, the preaponeurotic fat and orbital septum hang below the fusion line of the septum and aponeurosis. The hanging portion of the septum was dissected from the aponeurosis, plicated, and sutured to the dermis of the pretarsal flap. We performed the septodermal fixation technique in 512 patients over 3 years starting in March of 1992. Complications and changes of the folds were analyzed. The technique produced much less edema and discomfort and created more natural fold lines than any other technique. Patients were very lightly satisfied with the shape of the folds that we were able to design and adjust precisely with this method.

Surgical reconstruction of the contracted orbit.

Anophthalmic patients and patients afflicted with retinoblastoma incur severe deformity of the orbit. Treatment of the severely contracted orbit is very difficult, and patient satisfaction is often poor. Since 1988, we have performed temporalis muscle transfer and surgical expansion of the contracted bony orbit in 26 patients. Satisfactory results were obtained. Gradual expansion of the orbit was performed in case of congenital anophthalmic patients. The treatment should be established in multiplicity, among many methods available for contracted eye sockets, according to the degree of orbital deformity and the amount of residual conjunctiva. In case of severe deformity, volume expansion surgery and temporalis muscle transfer are necessary. If augmentation is required in the periorbital region, rib bone onlay graft must be performed. We were able to shorten the operative time by modifying the three-wall orbital expansion technique of Tessier and Wolfe to a more simplified method. Our observations show that our procedures achieved symmetry in both eyes in all patients, and there have been no remarkable complications.