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The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host’s cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2–6.4 ID50 g−1 was reduced to a remaining infectivity of 104.6–5.7 ID50 g−1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5–800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

Current anti-epidermal growth factor receptor (EGFR) therapy for oral cancer does not provide satisfactory efficacy due to drug resistance or reduced EGFR level. As an alternative candidate target for therapy, here we identified an oncogene, ROS1, as an important driver for oral squamous cell carcinoma (OSCC) metastasis. Among tumors from 188 oral cancer patients, upregulated ROS1 expression strongly correlated with metastasis to lung and lymph nodes. Mechanistic studies uncover that the activated ROS1 results from highly expressed ROS1 gene instead of gene rearrangement, a phenomenon distinct from other cancers. Our data further reveal a novel mechanism that reduced histone methyltransferase EZH2 leads to a lower trimethylation of histone H3 lysine 27 suppressive modification, relaxes chromatin, and promotes the accessibility of the transcription factor STAT1 to the enhancer and the intron regions of ROS1 target genes, CXCL1 and GLI1, for upregulating their expressions. Down-regulation of ROS1 in highly invasive OSCC cells, nevertheless, reduces cell proliferation and inhibits metastasis to lung in the tail-vein injection and the oral cavity xenograft models. Our findings highlight ROS1 as a candidate biomarker and therapeutic target for OSCC. Finally, we demonstrate that co-targeting of ROS1 and EGFR could potentially offer an effective oral cancer therapy.

Agriculture in the 21st Century is facing new challenges and transformation according to five general trends: commercialisation, internationalisation, science and technology, environment and energy production. In this laboratory, a series of research, development and commercialisation of biogas and enzyme technologies reflect these trends. Anaerobic digestion is a microbial process that converts organic waste into biogas, containing 60-70% methane. A simple thermophilic anaerobic digestion (TAnD) system for poultry manure was developed from the lab to the farm. TAnD at 50 to 60°C produces biogas at high rates and, as a result, the process is efficient and the digester is a compact unit. A pilot plug-flow TAnD was first constructed and operated at NCSU research farm. Multiple benefits of the system were demonstrated. Supported by UNDP, a TAnD to process five tons of manure daily (from 50,000 hens) was constructed in China and has been operating for 20 years. Large scale digesters processing hundreds of tons of manure daily are increasingly popular in China and Europe. Biogas from large digesters can be a significant source of energy to generate electricity or to fuel transportation. Beyond energy production, the digester is a rich source of biomaterials. A feather-degrading bacterium was discovered and isolated from TAnD, and its keratinase enzyme and the gene encoding for the enzyme were subsequently isolated. Genetic manipulation for over-expression and up-scale fermentation have now made the industrial production of the enzyme possible. As a heat-stable protease, the keratinase was found to be useful in processing feather meal, in enhancing feed digestibility and was able to degrade prion proteins (implicated as the disease vector in bovine spongiform encephalopathy).

SUMMARY Versazyme (VZ) is an approved keratinase-based feed additive produced naturally as a fermentation product of Bacillus licheniformis PWD-1. The experiment was designed to test the recovery and efficacy of VZ using typical feed industry pelleting parameters. The pelleted diets were conditioned to 85°C for 30 s and pelleted using a 4 × 32 mm die, cooled, and crumbled. A total of 192 male broiler chicks were fed a corn-soybean meal starter diet formulated at 18.5% CP to 21 d of age. The 4 treatments used in the feeding study were as follows: VZ 0.0%-mash, VZ 0.0%-pellet, VZ 0.1%-mash, and VZ 0.1%-pellet. Body weight and feed consumption were determined at 21 d of age. The recovery of VZ in feed was 93% in the mash diet and 66% in the pelleted diet. There was a 55-g increase in BW gain and 0.17 improvement of FCR when birds were supplemented with 0.1% VZ. Pelleting improved BW gain (P < 0.05) but did not significantly improve the FCR. Growth performance results indicate that pelleting did not affect the efficacy of VZ.

All-trans retinoic acid (ATRA) can induce acute respiratory distress syndrome in patients with acute promyelocytic leukaemia (APL). The current study investigated the role of monocyte chemotactic protein (MCP)-1 in the chemotactic transmigration of ATRA-treated NB4 (ATRA-NB4) APL cells toward A549 alveolar epithelial cells. NB4 and A549 cells were separately cultured with ATRA and/or dexamethasone (DEX). ATRA-NB4 cells were then placed in an upper insert and co-incubated with A549 cells or their conditioned medium (CM) located in a lower plate to test their transmigration activity. ATRA stimulated NB4 cells to transmigrate toward the A549 cells. The secretion of MCP-1 was enhanced by ATRA treatment in both A549 and NB4 cells. The binding assay demonstrated that ATRA-NB4 cells bound MCP-1. Pre-treatment of both CM-A549 cells with antibodies against MCP-1 and of ATRA-NB4 cells with antibodies against MCP-1 receptors reduced ATRA-NB4 cell transmigration. DEX did not suppress MCP-1 secretion and transmigration in ATRA-NB4 cells, although when applied to A549 cells, MCP-1 secretion was suppressed and ATRA-NB4 cell transmigration was attenuated. Monocyte chemotactic protein-1 secreted from alveolar epithelial cells plays an important role in the cell-cell interaction involved in the chemotactic transmigration of all-trans retinoic acid-treated acute promyelocytic leukaemia cells toward alveolar epithelial cells.

A preparation of keratinase (KE, 400,000 enzyme units/g) was evaluated as a feed additive for its effects on growth performance, nutrient digestibility and intestinal development in broiler chickens. Four experimental diets were arranged as a 2 × 2 factorial with two sources of protein, either soybean meal (SBM) alone or a combination of SBM and cottonseed meal (1:1 on crude protein basis) and with or without 1.0 g KE/kg diet. Dietary supplementation of KE increased body weight gain from 1 to 21 days of age and improved feed conversion ratio from 1 to 42 days of age. Also, KE supplementation enhanced nitrogen retention (P

A prion-like protein, Sup35NM-His6, which is safe, easy to produce and able to aggregate into stable amyloid, was developed. This study was to test the feasibility of using Sup35NM-His6 amyloid as a marker for standard sterilization methods known to be effective in inactivating infectious prions. Also Sup35NM-His6 aggregates were spiked into cow brain tissue homogenates to mimic prions in tissues to determine if the aggregate protein could be specifically detected by Western blot at the nanogram level. Like mammalian prions, the proteinase K (PK) resistant fractions of Sup35NM-His6 remain intact after autoclaving. Treatments with 0.1N NaOH or 1.0% NaOCl also resulted in PK undigestible residues. Exposure to the strong denaturant guanidine thiocyanate (>3 M) destabilized and made the protein PK-digestible. Under strong alkaline conditions of 1.0N NaOH, 2.5% NaOCl, or a combination of NaOH (0.1 and 1.0N) with autoclaving, Sup35NM-His6 was completely hydrolyzed such that it was no longer detectable by Western blot. Overall, these tests suggest that Sup35NM-His6 could be a useful tool to assess the effectiveness of prion degradation for the prevention of TSE.

Versazyme (VZ) is a keratinase-based feed additive. Broiler diets were formulated with low (95%), medium (100%), or high (105%) CP and amino acid levels relative to US Poultry Industry Reporting Service averages (2003). In each diet, amino acid minimums were maintained relative to the percentage of CP. The protein levels were as follows: starter—21, 22, 23%; grower—19, 20, 21%; finisher—17, 18, 19%; and withdrawal—16, 17, 18%. Mixed sex broilers 0 to 48 d of age were fed the 3 protein levels with and without VZ (0.10%) in a 3 × 2 factorial experiment. Statistical main effects indicated 48-d BW was greater on the high protein diets (2.42 kg) compared with low protein (2.36 kg), with the medium protein diet being intermediate (2.40 kg). Overall, VZ significantly (P

Recent studies indicate that enzymatic treatment of the infectious PrP Sc prion under defined conditions could be an effective method to inactivate infectious prions. However, field studies on prion inactivation are hampered by restricted access to the dangerous and expensive infectious prion material. Hence, a surrogate marker for infectious prions would facilitate more practical prion inactivation research. Protein Sup35p, a non-pathogenic prion-like protein produced in yeast, has physical and chemical properties very similar to the BSE prion. Sup35NM-His6, a derivative of Sup35p, was produced from Escherichia coli by gene cloning, protein expression and purification. Monomeric Sup35NM-His6 is soluble. When aggregated, it forms prion-like amyloid, insoluble and resistant to proteases. Similar to BSE prion, a pre-heating step renders this protein digestible by proteinase K, subtilisin and keratinase but not collagenase and elastase. These results indicated that Sup35NM-His6, being simple and inexpensive to produce and non-pathogenic, can be a potential ideal candidate of prion surrogate protein in the study of prion inactivation and prevention of prion diseases.

Aims: Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A sporulation-deficient mutant of B. licheniformis PWD-1, named B. licheniformis WBG, was developed and characterized. Methods and Results: The mutation was generated using the splicing by overlap extension PCR method (Gene SOEing) to create 256 bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise the nucleotide fragment 256 bp from the B. licheniformis chromosome. PCR analysis and DNA sequencing confirmed the spoIIAC gene deletion. Heat-treatment assays and electron microscopy verified the absence of spores. Conclusions: This asporogenic strain is able to express normal levels of keratinase when compared with its wild-type host. Significance and Impact of the Study: In this study, a method of constructing a stable sporulation-defective strain was developed. It can be potentially useful as a tool to generate asporogenic strains of Bacillus that retain their industrial capabilities for production of exoproteases and other exozymes.

A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.

Extracellular production of keratinase–streptavidin fusion protein (KER–STP) was accomplished by the cloning of Bacillus subtilis with a transforming plasmid carrying the kerA - stp fusion gene. The fusion protein was readily immobilized onto a biotinylated solid matrix by mixing in the culture medium. The properties and reaction kinetics of free and immobilized keratinase (KE) were characterized and compared. Heat stability and pH tolerance were greatly improved by immobilization, but the catalytic efficiency ( k cat / K m ) was reduced by eightfold. The yield of bio-immobilization using bioselective adsorption of the fusion protein was approximately 20%, as estimated from the activity of free KE. HPLC analysis of reaction products demonstrated the hydrolysis of feather keratin, casein, and bovine serum albumin (BSA) by the immobilized KE. The rates of reactions are lower than those of the free enzyme. On the other hand, the stability of the enzyme was greatly improved.

The objective of this study was to determine the efficacy of a broad-spectrum protease enzyme, PWD-1 keratinase, upon supplementation to corn-soy starter diets on growth performance of broiler chickens. Three experiments were conducted. In each experiment, 1-d-old broiler chicks were randomly assigned to 24 cage pens of eight birds per pen in a completely randomized design of five experimental treatments and grown to 21 or 26 d of age. Treatments in experiments 1 and 2 were control (C, 21.39% CP), low protein (LP, 18% CP), and LP supplemented with 0.05, 0.1, or 0.15% enzyme preparation (wt/wt). Treatments in experiment 3 were control (C), C+ 0.1% enzyme preparation (C+E) fed starting at either 1 or 5 d of age, LP and LP+ 0.1% enzyme preparation (LP+E). Feeding the LP+E diet produced numerically higher BW at 21 d of age (experiments 1 and 3) and a significantly higher BW at 26 d of age (experiment 2; 1,025 and 1,032 g vs. 965 g for 0.1 and 0.15% vs. LP, respectively, P < 0.05). Feed conversion ratio (FCR) was also improved when chicks were fed the LP+E diet both at 21 (experiment 3) and 26 d of age (experiment 2). In experiment 3, supplementing the C diets with 0.10% enzyme resulted in improvements (P < 0.05) in BW whether the enzyme was supplemented starting at 1 d (767 vs. 695 g for C+E vs. C, respectively) or 5 d of age (764 vs. 695 g for C+E vs. C, respectively). FCR was numerically improved. Furthermore, diets supplemented with the enzyme at any level resulted in reduction of jejunal viscosity at 22 and 27 d of age (P < 0.05). Results of these experiments indicate that the growth of broiler chickens can be significantly improved by dietary supplementation with PWD-1 keratinase.

Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase-streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24-28%, using both systems.

Fermentation scale-up was studied for the production of keratinase by Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6-10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37 degrees C for both strains, though B. licheniformis is thermophilic and grows best at 50 degrees C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37 degrees C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50 degrees C for growth followed by a shift to 37 degrees C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29.

The intensive and large-scale production of food animals and animal products has generated an enormous waste disposal problem for the animal industry. These wastes, which include animal excreta, mortalities, hair, feathers, and processing wastes, are largely organic materials and are convertible to useful resources. Making the conversion processes efficient and economical presents a great challenge to modern biotechnology. An efficient thermophilic anaerobic digester system has been developed that converts animal manure to methane for an energy source, solid residues for feed supplements, and liquid nutrients for aquaculture. This digester system also destroys pathogens and thus protects environmental health. During the development of this system, a feather-degrading bacterium was discovered and identified as a thermophilic Bacillus licheniformis, Strain PWD-1. The bacterium can ferment and convert feathers to feather-lysate, a digestible protein source for feed use. An enzyme, keratinase, secreted by this bacterium was purified and characterized. This keratinase is a potent protease that hydrolyzes all proteins tested, including collagen, elastin, and feather keratin. When the enzyme was mixed as an additive in feed, it significantly enhanced the digestibility of feather meal in chickens. In addition to feed technology, the bacterium and the enzyme are believed to have many other industrial and environmental applications.

The major cellular components of atherosclerotic lesions in several species have been shown to be smooth muscle cells (SMC) and macrophages. Many studies suggest the composition of a lesion varies depending on the stage of lesion development. For example, macrophages are believed to be involved in the initial events of fatty streak formation in many animals. This communication presents the first cellular study of quail atherosclerosis and demonstrates the alteration of cellular structure during the process of the disease in quail fed a cholesterol diet. Monoclonal antibodies to alpha-actin and chicken macrophages effectively identified the presence of SMC and macrophages, respectively, as constituents of the atherosclerotic lesions. Macrophage presence, as well as SMC proliferation, was observed in early lesions. Although the first cell type to be involved in the initial stages of atherogenesis cannot be defined, the results suggest early intervention of macrophages and SMC. Scanning electron microscopic examination of the aortic arch demonstrates the obvious differences in appearance of the endothelial surface of normal and diseased quail. The accumulation of subendothelial foam cells causes the lumen surface to bulge irregularly into the lumen. The results of the present study are important to the evaluation of the key cellular events of atherogenesis.

Feeding trials were designed to evaluate the nutritive value of feathers treated by a feather-degrading bacterium, Bacillus licheniformis (Strain PWD-1). An initial experiment compared feathers that had been treated under aerobic or anaerobic conditions with untreated feathers as 25% of the feed protein in the diet. The test diets were fed to growing broiler chicks from Day 6 through Day 21 posthatch. The anaerobically fermented product, feather-lysate, resulted in improved growth responses of 6.9 and 19.3% over the aerobic- and nonbacterial-treated feather feed products, respectively, but 6.4% less than a standard com-soybean-based diet In a subsequent experiment, 3 or 6% of feather-lysate, untreated feathers, and commercial feather meal were fed to chicks on a basal diet containing only 10% protein. The linear growth response slopes were compared with the slope resulting when chicks were fed diets containing soybean protein in graded levels from 10 to 19% of the total feed. Feather-lysate supplemented with lysine, methionine, and histidine produced a growth curve identical to that of soybean meal. Commercial feather meal supported an improved growth rate over the untreated feathers but significantly less than feather-lysate. These results indicate that the anaerobic fermentation of feathers offers a potential new process for feather waste treatment to provide a nutritious feed protein.

Extracts of snake venom have been widely used for the treatment of vascular thrombotic diseases, yet the therapeutic mechanism is not clear. The effect of snake venom fractions on atherosclerosis in Japanese quail was studied. The venom of Agkistrodon halys was fractionated by DEAE-cellulose chromatography and the pooled protein fractions that resulted were injected intravenously into the quail with aortic atherosclerosis induced by dietary cholesterol. After 7 weeks of injections on every other day, the quail were killed, blood clotting times and serum cholesterol levels were determined, and aortic atherosclerosis and fatty liver were scored. The results showed that while no regression of atherosclerosis was observed, the lowering of serum cholesterol, prolonged blood clotting time and reduced fatty liver were significantly affected by the injection of one of the pooled protein fractions. This venom fraction contained two major protein components, one of which had arginine esterase activity. From this study we conclude that snake venom has little effect on the regression of atherosclerosis, but it prolongs blood clotting and lowers serum cholesterol.

A bio-process called thermophilic anaerobic digestion that converts poultry waste into biogas energy was developed in our laboratory. During operation, feathers in the waste were completely degraded. This observation prompted a series of studies that have led to new technology and commercial developments. A feather-degrading bacterium was discovered and isolated. An enzyme called keratinase capable of hydrolysing the feather protein keratin was purified. The gene encoding this enzyme was isolated and sequenced. Genetic modification enabled the over expression of the gene in the parent strain as well as in other Bacillus strains. Scale-up fermentation, first in 150 l and then in 50 kl fermentors, made the mass production of the enzyme possible. Subsequently, in application research, keratinase was shown to partially hydrolyse feathers and convert them into a digestible feed protein. When supplemented as a feed additive on standard diet, the enzyme promoted chicken growth by improving dietary protein digestibility. Ifcommercially developed, keratinase can lower feed cost, increase poultry meat yield, and upgrade the utilization of less digestible protein feedstuffs. From an environmental point of view, keratinase has the potential to reduce waste nitrogen excretion by farm animals. More recently, keratinase was found to be able to degrade prion protein that is believed to cause transmissible spongiform encephalopathies (TSE) including ‘mad cow disease’, sheep scrapie, deer chronic wasting disease and human Creutzfeldt–Jakob disease. This discovery indicated the potential use of keratinase in a new enzymatic process to destroy prion protein and possibly prevent TSE, in a cost-effective and environmentally friendly manner. To test and to develop the enzymatic process, a prion-like surrogate protein (PSP) has been developed as a safe bio-marker. The combination of keratinase and PSP can provide a useful toolbox for TSE prevention. Based on the keratinase platform technology and its applications, BioResource International (BRI), a biotechnology company, was founded to serve the poultry and animal industries.

Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.

Day-old male broiler chicks were randomly assigned to 32 floor pens in a completely randomized block design and grown to 6 wk of age. Birds in experiment 1 were fed 1 of 2 basal diets supplemented with or without a protease containing feed additive, Versazyme (VZ). The 4 treatments were 1) control (C), a corn-soybean meal diet that contained 95% of amino acids recommended by NRC except for threonine and isoleucine; 2) C + 0.1% VZ (wt/wt) (C+) in the starter diet only; 3) high (HP) amino acid diet, a corn-soybean meal diet with 100 to 105% of amino acid recommended by NRC except for threonine and isoleucine; and 4) HP + 0.1% VZ (wt/wt) (HP+) in starter diet only. Supplementing both diets with VZ improved BW and feed conversion ratio (FCR) at 21 d of age and BW at 42 d of age. Cumulative 42-d FCR was only improved in birds fed the HP+ diet. Birds in experiment 2 received the following treatments: 1) HP, 2) HP + 0.1% VZ batch A (wt/wt) (A) in starter diet only, and 3) HP + 0.1% VZ batch B (wt/wt) (B) in starter diet only. Enzyme supplementation improved 22-d BW and FCR. There was no significant difference in BW at 43 d of age. Both A and B improved overall FCR (1.758 and 1.751 vs. 1.79 for A and B vs. HP, respectively). In conclusion, supplementation of starter broiler diets with VZ resulted in improved market growth performance.

Keratinase isolated from Bacillus licheniformis PWD-1 was immobilized on controlled-pore glass beads. The immobilized keratinase demonstrated proteolytic activities against both insoluble feather keratin and soluble casein. It also displayed a higher level of heat stability and an increased tolerance toward acidic pHs compared with the free keratinase. During a continuous reaction at 50(deg)C, the immobilized keratinase retained 40% of the original enzyme activity after 7 days. The immobilized keratinase exhibits improved stability, thereby increasing its potential for use in numerous applications.

To increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed. Integrative vectors carrying kerA with or without P43-promoter were constructed and subcloned into B. licheniformis T399D and Bacillus subtilis DB104. In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot. The optimal integration of kerA was found in the range of 3-5 copies. Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase. In DB104, kerA was cloned in the plasmid, not integrated into the chromosome. The strong constitutive promoter P43 not only increased the keratinase production in plasmid-based expression in DB104 but also improved the enzyme yield of the integrants of T399D. New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate. When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4-6 times.

Prions-infectious agents involved in transmissible spongiform encephalopathies-normally survive proteolytic and mild protein-destructive processes. Using bacterial keratinase produced by Bacillus licheniformis strain PWD-1, we tested conditions to accomplish the full degradation of prion protein (PrP) in brain-stem tissue from animals with bovine spongiform encephalopathy and scrapie. The detection of PrPSc, the disease-associated isoform of PrP, in homogenates was done by Western blotting and various antibodies. The results indicated that only in the presence of detergents did heat pretreatment at >100 degrees C allow the extensive enzymatic breakdown of PrPSc to a state where it is immunochemically undetectable. Proteinase K and 2 other subtilisin proteases, but not trypsin and pepsin, were also effective. This enzymatic process could lead to the development of a method for the decontamination of medical and laboratory equipment. The ultimate effectiveness of this method of prion inactivation has to be tested in mouse bioassays.

The kerA gene which encodes the enzyme keratinase was isolated from the feather-degrading bacterium Bacillus licheniformis PWD-1. The entire gene, including pre-, pro- and mature protein regions, was cloned with Pker, its own promoter, P43, the vegetative growth promoter, or the combination of P43-Pker into plasmid pUB18. Transformation of the protease-deficient strain B. subtilis DB104 with these plasmids generated transformant strains FDB-3, FDB-108 and FDB-29 respectively. All transformants expressed active keratinase in both feather and LB media, in contrast to PWD-1, in which kerA was repressed when grown in LB medium. With P43-Pker upstream of kerA, FDB-29 displayed the highest activity in feather medium. Production of keratinase in PWD-1 and transformants was further characterized when glucose or casamino acids were supplemented into the feather medium. These studies help understand the regulation of kerA expression and, in the long run, can help strain development and medium conditioning for the production of this industrially important keratinase.

Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers. By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease. The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined. A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA. Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers. PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions. The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B. licheniformis NCIMB 6816. The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria. The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases. Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media.

Much progress has been made in last two decades in research, prevention, and treatment of atherosclerosis and subsequent coronary heart diseases. Hypotheses have been proposed to define a unifying concept for the basic mechanisms of atherogenesis. The response-to-injury hypothesis, as recently updated (Ross, 1993a,b), suggests that various forms of insults to the endothelium and arterial cells initiate a chronic, inflammatory response featuring the subendothelial infiltration and activation of macrophages and T-cells. Upregulation of expression and secretion of cytokines and growth factors associated with these activated leukocytes result in smooth muscle cell (SMC) migration and proliferation into the intima of the arterial wall. Simultaneous with this process is thought to be the modification of lipoproteins in the arterial wall. In one such process low-density lipoproteins (LDL) can be oxidatively modified and taken up by macrophages to become foam cells (Steinberg et al., 1989). Because it is cytotoxic, oxidized LDL may kill macrophages and endothelial cells. In response to endothelial injury, platelets adhere, aggregate and secrete platelet-derived growth factor (PDGF), which further stimulates the proliferation of SMCs and thrombosis as proposed in the original response-to-injury hypothesis (Ross and Glomset, 1976).

Proliferation of aortic smooth muscle cells (SMCs), an important feature of atherosclerosis, occurs in the early stage of the disease in Japanese quail when fed an atherogenic diet. Quail aortic SMCs were isolated from a total of 32 quail and cultured in Eagle's minimum essential medium supplemented with 10% bovine fetal serum. Studies were carried out to characterize the cells in vitro. They grew actively in the early passages, but began to develop cytopathic changes after 3-4 passages and eventually cell death. The pathobiology had similarities to virus infected cells. Whole-mount electron microscopic examination detected virus-like particles in the cultured cells. All cell lines tested were positive for viral infection as screened by DNA dot blot hybridization using a genomic library of Marek's disease herpesvirus as a probe. However, the attempt to isolate the virus was unsuccessful. In summary, these cell lines are believed to be infected by a putative quail herpesvirus. Results of this in vitro cellular study support previous findings that the atherosclerosis susceptible quail are latently infected in vivo. Future studies will be needed to determine whether the virus plays a role in the pathogenesis of atherosclerosis in this animal model.

A keratinase was isolated from the culture medium of feather-degrading Bacillus licheniformis PWD-1 by use of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchange and Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purified keratinase is monomeric and has a molecular mass of 33 kDa. The optimum pH and the pI were determined to be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was 50°C. The enzyme is stable when stored at −20°C. The purified keratinase hydrolyzes a broad range of substrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase is a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal.

A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to grow with feathers as its primary source of carbon, sulfur, and energy. Subsequently, a feather-hydrolytic, endospore-forming, motile, rod-shaped bacterium was isolated from the feather-degrading culture. The organism was Gram stain variable and catalase positive and demonstrated facultative growth at thermophilic temperatures. The optimum rate of growth in nutrient broth occurred at 45 to 50°C and at pH 7.5. Electron microscopy of the isolate showed internal crystals. The microorganism was identified as Bacillus licheniformis PWD-1. Growth on hammer-milled-feather medium of various substrate concentrations was determined by plate colony count. Maximum growth (approximately 10 9 cells per ml) at 50°C occurred 5 days postinoculation on 1% feather substrate. Feather hydrolysis was evidenced as free amino acids produced in the medium. The most efficient conditions for feather fermentation occurred during the incubation of 1 part feathers to 2 parts B. licheniformis PWD-1 culture (10 7 cells per ml) for 6 days at 50°C. These data indicate a potential biotechnique for degradation and utilization of feather keratin.

The effect of anaerobic digestion of poultry waste on oocysts of the protozoan Eimeria tenella, a common enteric pathogen that causes coccidiosis in poultry, was investigated in this study. Thermophilic (50 degrees C) and mesophilic (35 degrees C) anaerobic digestors, with poultry manure as the substrate, were inoculated with the oocysts. The oocysts were damaged during anaerobic digestion, as determined by morphological change and loss of their ability to sporulate. The recovered oocysts were tested for their infectivity in young chicks, as measured by body weight gain, mortality, and cecal lesions. Oocysts lost all their infectivity during thermophilic digestion, while oocysts subjected to mesophilic digestion remained moderately infective in comparison with untreated oocysts, which produced severe coccidiosis, high mortality, and low body weight gain in chicks. Oocysts were inactivated at 50 degrees C when they were suspended in digestor fluid or saline. Inactivation at 35 degrees C was significantly stronger in the digestor fluid than in the saline, which implied that factors other than temperature were involved in the lethal effect of anaerobic digestion on protozoan oocysts. In this study we demonstrated that the treatment of animal waste by anaerobic digestion, especially at a thermophilic temperature, has the benefits of pathogen control and protection of human and animal health in a farm environment.

Methane-producing, cellulolytic, feather-degrading, and total anaerobic microbial populations were enumerated in four laboratory-scale (l l) thermophilic (50°C) poultry waste digesters over a 40d period. Four different operation conditions were: 5 d retention time (RT), 6% volatile solids (VS); 5 d RT, 3% VS; 10 d RT, 6% VS; and 10 d RT, 3% VS. Laying hen manure was the sole source of substrate and micro-organisms. At theoretical steady state (day 40) the biogas volumetric rate was near 3.0 l/l digester volume (l/l/d) in all but the 10 d RT, 3% VS digester which was 2 l/l/d. The total viable anaerobic population was > 106 cfu/ml digester fluid at the first sampling and stabilized at 107–108 cfu/ml between days 20 and 40 in all digesters. Methane-producing bacteria increased from ≤ 10/ml early in the sampling period to 105/ml at steady state in all but the 5 d RT, 3% VS digester which was highest at 107/ml. Cellulolytic micro-organisms were low throughout the 40 d, generally less than 10/ml. Feather-degrading micro-organisms ranged from near 102–105 at steady state and were decreasing in number near day 40 in all but the 10 d RT, 6% VS digester which maintained 105/ml after day 20. A feather-degrading culture was enriched from this digester and subsequently adapted to grow in a medium with feather as the sole source of carbon. Results of this study provide information regarding potential biological upgrading of poultry waste digesters for increased operational efficiency and potential industrial application of a feather-hydrolytic micro-organism.

A poultry waste digester was constructed at the North Carolina State University Poultry Research Farm to produce methane from the waste of caged layer hens. A solid by-product (SBP) from the digester was recovered and dried for chemical and biological analysis as a feed supplement. Dried SBP was chemically analyzed for minerals, trace elements, and certain vitamins. The SBP was evaluated as a phosphorus (P) supplement in broiler diets. Biological availability of P in the SBP was determined by a slope ratio assay with femur bone ash as the criterion of P availability. Dietary treatments consisted of graded levels of standard available P (AP) supplemented as to provide .2, .3, .4, and .6% AP and graded levels of SBP added to provide 1.5, 2.5, 3.5, and 4.5% SBP to a corn-soy basal diet. Two treatments, containing 1.5% and 2.5% SBP added to an adequate diet (.4% AP) were used to determine if other factors in SBP were contributing to femur bone ash. The P in SBP was determined to be 90% available to the growing broiler chick. Femur bone ash did not respond to SBP in the diets already adequate in AP. Body weight gain, feed consumption, and feed conversion were not significantly different (P less than .05) among treatments with similar AP levels. The study concluded that SBP is a safe, effective feed supplement that supplies P, Ca, and total minerals.

Gas chromatography of the methyl esters of lipoic (1,2-dithiolane-3-pentanoic), bisnor -lipoic (1,2-dithiolane-3-propanoic), tetranor -lipoic (1,2-dithiolane-3-carboxylic) acids, and their reduced (dithiol) forms was carried out on a Poly A-103 column. This new chromatographic method is sensitive and adaptable to determine lipoic acid content in animal tissues and other biological materials. Livers from 1-week-old chicks and eggs at different incubation times were pooled, lyophilized, delipidated, hydrolyzed, and extracted. The extracts, after methylation, were chromatographed and quantitated for lipoate. Overall recovery using [ 14 C]lipoic acid was found to be 34%. The corrected level of lipoic acid in the chick liver was in the range 5–10 μg/g. The level in the egg increased from 1–2 μg/egg at 0 day to 22–24 μg/egg at 17 days of incubation.

A new animal model of atherosclerosis has been developed through genetic selection of Japanese quail ( Coturnix coturnix japonica ) into susceptible (SUS) and resistant (RES) lines. Characterization of the selected quail has shown that the RES birds were resistant to the disease and developed little atherosclerosis on a diet containing 1% cholesterol. The SUS birds were sensitive and developed severe atherosclerosis in 8–9 wks on a diet containing only 0.5% cholesterol. The histology of the progression of atherosclerosis in the SUS quail was studied. It bore a morphological similarity to that of human arterial atherosclerosis. The atherosclerotic plaque was characterized by intimal thickening, the presence of foam cells, the proliferation of smooth muscle cells and/or fibroblasts, and the formation of scar with collagen deposition. We believe that these two lines of quail may serve as a valid animal model for the study of the genetic and biochemical basis of cholesterol-induced atherosclerosis.

This study was undertaken to determine the influence of levels of soy protein on cholesterol metabolism and the development of atherosclerosis in Japanese quail (Coturnix coturnix japonica). Quail were fed purified diets containing one of four levels (10, 20, 40 or 60%) of soy protein either with (atherogenic) or without (control) 0.5% cholesterol. Results showed that higher proportions of protein (40 and 60%) in atherogenic diets had a preventive effect on the development of atherosclerosis in adult male quail. For the atherogenic diets the higher protein levels resulted in significantly lower serum and aorta cholesterol concentrations than observed with the 10 or 20% protein levels and free of aortic atherosclerosis. A dual isotope technique was used to measure the absorption rates for cholesterol in quail fed with 20 and 40% soy protein. Absorption rates were not affected by the level of dietary protein but were influenced by the presence of dietary cholesterol. Relative rates were 40% in quail fed control diets and 30% in quail fed atherogenic diets. The excretion of neutral sterols, largely cholesterol, and bile acids increased with the high intake of soy protein. These results demonstrate that in quail the presence of higher dietary levels of soy protein has both a hypocholesterolemic action and a preventive effect on cholesterol-induced atherosclerosis and one of the possible mechanisms is through increased excretion of cholesterol.

Japanese quail genetically selected on the basis of atherosclerosis susceptibility were tested for infection by Marek's disease herpesvirus (MDV). Viral DNA was detected in the atherosclerotic aortas of susceptible (SUS) quail by the technique of DNA hybridization. Southern blot analysis demonstrated that restriction mapping of aortic DNA was specific and different from that of MDV. Screening of quail embryos by dot-blot hybridization detected that MDV DNA existed in 100% of SUS quail tested. Resistant (RES) quail were a mixed population, with 16% of embryos resembling the SUS group. Functional MDV was not found by a number of methods including virus isolation, serological test, and exposure of sentinel chicks to SUS quail. These results suggest that the SUS quail possess a portion of the MDV genome in the germline, and the viral genes have been coselected by their susceptibility to cholesterol-induced atherosclerosis.

SUMMARY: Pseudomonas putida LP, which grows on lipoate, NH4NO3 and mineral salts, converts most of the organic substrate to bisnor-lipoate (1,2-dithiolane-3-propanoic acid) and acetyl-CoA. D-, L-, or DL-lipoate serve equally well as carbon and sulphur sources. There was no growth on or bacterial oxidation of the chemically synthesized bisnor- or tetranor-(1,2-dithiolane-3-carboxylic acid) chain-shortened analogues, but these, as well as lipoate, could supply the sulphur needed for growth when acetate was provided as the sole source of carbon. The uptake of lipoate by the bacterium is very slow and non-inducible, while the uptake of acetate is faster than octanoate. The oxidation of octanoate is more rapid and extensive than that of lipoate. Levels of acyl-CoA synthetase are not affected by the source of carbon, but activities of isocitrate lyase and malate synthase are higher when the cells are grown in acetate, octanoate or lipoate and lower when glucose is the carbon source. The glyoxylate cycle is induced to facilitate utilization of acetyl-CoA derived from lipoate, which is also degraded to water-soluble catabolites that yield the much smaller amount of sulphur required for growth.

A new method is described for determination of antitrypsin activity based on inhibition of trypsin solubilization of calcium caseinate in agar plates. The method was applied to analyze soybeans after graded heat treatments for their antitrypsin content. Biological determination of protein and energy values of the soybean samples showed direct correlation of these values with the destruction of antitrypsin as measured by the new method, using rainbow trout.

Two lines of Japanese quail were genetically selected. One is highly susceptible (SUS) to atherosclerosis, developing severe aortic atherosclerosis in 9 weeks by feeding 0.5% cholesterol in the diet. The other is highly resistant (RES), developing little disease under the same conditions. The pathology of quail atherosclerosis, characterized by intimal thickening, the presence of foam cells, the proliferation of myofibroblast cells, and the formation of scar with collagen deposition, is similar to the human disease. Because of its small size, low feed consumption, and short life cycle, these quail are an excellent model for the study for prevention and pathogenesis of atherosclerosis. A study has shown that the quail respond to treatment with nicotinic acid, gemfibrozil, clofibrate, and aspirin with reduction of atherosclerosis. More recently these birds were tested for the presence of a herpes virus, Marek disease virus (MDV), as a possible etiologic agent. Initial diagnosis by isolation of MDV and agar precipitin test was negative. However, when a gene library of MDV was used to prepare a battery of radioactive DNA probes, high-stringency DNA hybridization has demonstrated the presence of viral genes in quail aorta, heart, nucleated RBC, and embryo. The intensity of hybridization increased with the increased severity of atherosclerosis in aortas. In embryos, all SUS quail were positive, but only a part of RES quail were positive. These results indicated that these quail were latently infected by a putative herpes virus closely related to MDV. Since DNA homology was detected in the embryo, it is possible that the viral genes are integrated in the host DNA, and they are the heritable elements for the susceptibility to atherosclerosis.

A strain of bacteria that can degrade lipoic acid was isolated from soil. The bacterium, adapted to use 0.4% dl -lipoate as the sole organic substrate to supply carbon, sulfur, and energy, was identified morphologically and physiologically as a strain of Pseudomonas putida . Degradation of 1,6 - 14 C -lipoic acid, synthesized from 1,6 - 14 C -adipic acid, was evidenced by: (i) loss of approximately 50% of the total radioactivity from the medium after bacterial growth; (ii) appearance of 14 C-degradation products upon paper and thin-layer chromatography of the culture medium; and (iii) oxygraphically measured utilization of O 2 by cells in the presence of lipoate or other oxidizable substrates. Analyses of the benzene extract of culture medium by infrared, nuclear magnetic resonance, and mass spectrometry, and by gas-liquid chromatography after desulfuration, have characterized bisnorlipoic acid, or 4,6-dithiohexanoic acid, as the major catabolite present in the medium. β-Oxidation of the side chain is thus proven to be a pathway employed by the pseudomonad to degrade lipoic acid.