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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.

Ankyloglossia is a failure of the tongue to release from the oral floor with reported consequences that include breastfeeding difficulties and speech impediments. Frenotomy is a commonly performed procedure for the treatment of ankyloglossia. Laser (light amplification by stimulated emission of radiation) is one of several mediums used to perform frenotomies. Although most frenotomies are uncomplicated, there remains a small possibility of complication, such as infection, pain, ductal injury, and hemorrhage, even in expert hands. Because frenotomies are most often performed in infants, postoperative hemorrhage is an important complication to look for as even small amount of bleed may prove fatal, due to low blood volume reserve. We report a case of delayed hemorrhage after laser frenotomy in a 6-week old infant displaying shock symptoms and required fluid resuscitation.

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.

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 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.

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.

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.

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.

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.

Background: The Janus kinase-2 (JAK-2) V617F mutation has been recently reported in patients with myeloproliferative disorders (MPD), which is believed to underlie growth factor hypersensitivity displayed by haematopoietic progenitors in these disorders. However, its frequency has been rarely determined in Taiwanese patients. Methods: The frequency of JAK2-V617F mutation in patients with polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis (IMF) was determined in the DNA from the peripheral blood leucocytes of 108 patients by genomic polymerase chain reaction and restriction enzyme-based assay. Results: The JAK2-V617F mutation could be detected in 28 of 33 polycythaemia vera patients (85%), 29 of 49 essential thrombocythaemia patients (59%) and 2 of 6 IMF patients (33%), but was not detected in 11 patients with myelodysplastic syndrome or another 10 with other haematological diseases. The presence of the JAK2 mutation was associated with specific MPD disease subtypes (P = 0.007), longer disease duration (P = 0.005), splenomegaly (P = 0.019), a higher white blood cell count (P = 0.002) and a higher haemoglobin level (P = 0.036). However, the overall risk of thrombosis or bleeding was not affected by the presence of the JAK2 mutation (32 vs 17%; P = 0.22). Conclusion: The JAK2-V617F mutation can be frequently detected in the Taiwanese patients with MPD disorders and therefore should be incorporated into the initial evaluation of patients suspected of MPD.

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.

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.

Objectives: To investigate and compare closed and open revision techniques in the treatment of ununited femoral shaft fractures associated with locked nail breakage. Design: Retrospective. Setting: University hospital. Methods: Ununited femoral shaft fractures associated with locked nail breakage were treated with either closed or open revision (nine or eighteen cases, respectively), The closed technique entailed closed removal of the broken nail and reinsertion of a stable intramedullary nail after reaming the marrow cavity The open technique included open removal of the broken nail, reinsertion of a stable intramedullary nail or plate, and cancellous bone graft supplementation. Union rate, union period, perioperative course, and complications were compared. Results: Eight closed and fifteen open technique cases were followed for at least one year (median two years), Cases treated with the closed technique had a union rate of 100 percent, a union period of 4.4 ± 0.9 months, an operating time of 1.5 ± 0.4 hours, no blood transfusion, and no complications. Open technique cases demonstrated a union rate of 100 percent, a union period of 5.7 ± 1.5 months (p = 0.033), an operating time of 2.4 ± 0.4 hours (p < 0.001), blood transfusion of 1,000 ± 500 milliliters (p < 0.001), and no complications. Conclusions: We recommend the closed revision technique because its union period and operating time are shorter, and it does not require a blood transfusion. Because there is no local wound dissection, infection rates should also be lower. However, the procedure is technically demanding. If it cannot be completed successfully, using the open technique can still achieve a satisfactory outcome.

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.

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.

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.

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.

Nutritional muscular dystrophy in the chick results from the simultaneous deficiency of vitamin E and cystine. Being a biological antioxidant, vitamin E might be functional in maintaining a proper redox state of the sulfur-containing amino acid in the proteins. The analyses of protein-bound sulfhydryls and disulfides at onset of muscular dystrophy in young chicks were carried out. The ratio of disulfide to sulfhydryls increased two- to three-fold in dystrophic muscle as compared to that in the control muscle proteins. Dystrophic and normal muscle proteins also were subjected to SDS-gel electrophoresis. Proteins of low molecular weight, supposedly derived from proteolysis, were present in the gels of the dystrophic muscle and absent in those of normal muscle extracts. As a result of these studies, a chemical model has been proposed to explain the oxidative deterioration of proteins in nutritional muscular dystrophy due to vitamin E deficiency.

A selenium deficiency in chicks produces degeneration and fibrosis of the pancreas. An investigation was undertaken to determine whether or not the activity of lipoic acid is impaired in the pancreas of selenium-deficient chicks. Enzymatic analyses of selenium-deficient chick tissues showed a reduction in lipoamide dehydrogenase both in pancreas and liver at a very early stage of growth. Using both direct measurements and sucrose-gradient cellular fractionation, it was found that the lipoamide dehydrogenase activity of the supernatant increased, and that of the mitochondria decreased in selenium-deficient as compared to normal chick livers. These results indicate an increased fragility of the mitochondrial membranes in selenium-deficient chicks.

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.