A double-cos-site vector containing a multiple cloning site flanked by T7 and T3 RNA polymerase promoters.

A double-cos-site cosmid vector, c2XMCS, contains a multiple cloning site (MCS) with fifteen restriction sites flanked by phage T7 and T3 RNA polymerase promoters. The two cos sites in the cosmid vector enable the construction of cosmid libraries from unfractionated partial digests of genomic DNA. This simple construct combines the efficiency of a double-cos-site cosmid with the versatility provided by the MCS from pBluescript.

Consumption of flavonoids in onions and black tea: lack of effect on F2-isoprostanes and autoantibodies to oxidized LDL in healthy humans.

BACKGROUND: Oxidative damage to lipids in vivo may be involved in the development of atherosclerosis and cancer. Onions and black tea are foods rich in flavonoids, predominantly the flavonoid quercetin, which is a potent in vitro inhibitor of membrane lipid peroxidation and LDL oxidation. OBJECTIVE: Our objective was to investigate the effects of consuming a high-flavonoid (HF) diet enriched with onions and black tea on indexes of oxidative damage in vivo compared with a low-flavonoid (LF) diet. DESIGN: Thirty-two healthy humans were studied in a randomized crossover design. Indexes of oxidative damage used were plasma F2-isoprostanes (a biomarker of lipid peroxidation in vivo) and the titer of antibodies to malondialdehyde (MDA)-modified LDL. RESULTS: There were no significant differences in the intake of macronutrients or assessed micronutrients, plasma F2-isoprostane concentrations, and MDA-LDL autoantibody titer between the HF and LF dietary treatments. In the men, however, plasma concentrations of the F2-isoprostane 8-epi-prostaglandin F2alpha were slightly higher after the HF treatment phase than after the LF treatment [0.31 +/- 0.029 nmol/L (111 +/- 10.4 ng/L) compared with 0.26 +/- 0.022 nmol/L (92 +/- 7.8 ng/L); P = 0.041]. In all subjects, plasma quercetin concentrations were significantly higher after the HF treatment phase than after the LF treatment: 221.6 +/- 37.4 nmol/L compared with less than the limit of detection of 66.2 nmol/L. CONCLUSION: Flavonoid consumption in onions and tea had no significant effect on plasma F2-isoprostane concentrations and MDA-LDL autoantibody titer in this study and thus does not seem to inhibit lipid peroxidation in humans.

Isoflavone phytoestrogens consumed in soy decrease F(2)-isoprostane concentrations and increase resistance of low-density lipoprotein to oxidation in humans.

BACKGROUND: Oxidative damage to lipids may be involved in the etiology of atherosclerosis, cardiovascular disease in general, and cancer. The soy isoflavone phytoestrogens, genistein and daidzein, and equol (a daidzein metabolite produced by intestinal microflora) are antioxidants in vitro; equol is a particularly good inhibitor of LDL oxidation and membrane lipid peroxidation. OBJECTIVE: We sought to investigate the effects of a diet enriched with soy containing isoflavones on in vivo biomarkers of lipid peroxidation and resistance of LDL to oxidation, compared with a diet enriched with soy from which the isoflavones had been extracted. DESIGN: : A randomized, crossover design was used to compare diets enriched with soy that was low or high in isoflavones in 24 subjects. Plasma concentrations of an F(2)-isoprostane, 8-epi-prostaglandin F(2)(alpha) (8-epi-PGF(2)(alpha)), a biomarker of in vivo lipid peroxidation, and resistance of LDL to copper-ion-induced oxidation were determined. RESULTS: Plasma concentrations of 8-epi-PGF(2)(alpha) were significantly lower after the high-isoflavone dietary treatment than after the low-isoflavone dietary treatment (326 +/- 32 and 405 +/- 50 ng/L, respectively; P = 0.028) and the lag time for copper-ion-induced LDL oxidation was longer (48 +/- 2.4 and 44 +/- 1.9 min, respectively; P = 0.017). Lag time for oxidation of unfractionated plasma and plasma concentrations of malondialdehyde, LDL alpha-tocopherol, polyunsaturated fatty acids, and isoflavonoids did not differ significantly between dietary treatments. CONCLUSIONS: Consumption of soy containing naturally occurring amounts of isoflavone phytoestrogens reduced lipid peroxidation in vivo and increased the resistance of LDL to oxidation. This antioxidant action may be significant with regard to risk of atherosclerosis, cardiovascular disease in general, and cancer.

Construction of portable intron cassettes for the delivery and expression of foreign genes.

The use of viral vectors to deliver foreign genes offers some promise of generating new and more efficacious vaccines. However, the insertion of foreign genes into viral genomes often results in the insertional mutagenesis of one or more genes that adversely affect replication. In an attempt to overcome this problem, we constructed two portable intron cassettes. The cassettes were derived from the adenovirus late leader 1 intron and were cloned into either the chloramphenicol acetyltransferase (CAT) gene or the LacZ gene of Escherichia coli. The intron cassettes were transfected into chicken embryo fibroblasts (CEFs) and the cell lysates were later assayed for either beta-galactosidase (beta-Gal) or CAT activity. The first intron cassette (type A) contained flanking adenovirus exon sequences. Consequently, the flanking adenovirus exon sequences remained in the spliced transcript. With the type A intron inserted in the correct orientation for splicing, CAT activity was not diminished. However, in the reverse orientation, no CAT activity could be detected. The second intron cassette (type B) had the splice donor and splice acceptor sites converted to the blunt-end restriction endonuclease sites Pml I and Pvu II, respectively. The blunt-end restriction endonuclease sites enabled the portable intron to be removed from the flanking adenovirus exon sequences and inserted into any blunt-end restriction endonuclease site in the recipient gene. After splicing, no adenovirus exon sequences remained in the recipient gene's RNA transcript. To demonstrate its usefulness, an insertion cassette was made by cloning the E. coli LacZ gene into a multiple cloning site within the type B intron. The insertion cassette was then cloned into a Pvu II site in the middle of the CAT gene. Following transfection in CEFs, high levels of both CAT and beta-Gal were detected, demonstrating that both genes were properly transcribed and translated.

Transcription vectors that facilitate the identification and mapping of RNA splice sites in genomic DNA.

Two transcription vectors were constructed that can identify the splice sites at exon-intron boundaries of inserted DNA fragments possessing the complementary splice site. One vector contains the 5' splice donor site and flanking exon-intron sequences from the 3' end of the adenovirus first late leader. The other vector contains the 3' splice acceptor site and the branch acceptor site, plus the flanking exon-intron sequences from the 5' end of the adenovirus second late leader. Both vectors contain a multiple cloning site for insertion of DNA fragments. DNA fragments supplying the complementary splice site, including the adjacent exon and intron sequences, were inserted into the vectors. The vectors were used as templates for the synthesis of chimeric RNA transcripts that were spliced in in vitro splicing extracts. Chimeric transcripts from the vectors containing complementary splice site boundary regions from the human growth hormone gene were accurately spliced in vitro. A splice site from a human growth hormone intron that is not normally spliced in vitro was spliced when paired with an adenovirus splice site. These vectors can be used to identify splice sites and to determine the lengths of exons and their attached introns within a DNA fragment of unknown coding content.

Cosmid library of the turkey herpesvirus genome constructed from nanogram quantities of viral DNA associated with an excess of cellular DNA.

A protocol was designed for the rapid and efficient construction of cosmid libraries from cell-associated viral genomes available in very low quantities. Purification of viral DNA from cellular DNA was unnecessary. The vast excess of cellular DNA compensated for the limited amount of available viral DNA, enabling titration of the restriction endonuclease partial digest. A cosmid library of the turkey herpesvirus DNA genome was constructed from 1.5 micrograms of cellular DNA containing approximately 6 nanograms of viral DNA.

Flavonoids protect against oxidative damage to LDL in vitro: use in selection of a flavonoid rich diet and relevance to LDL oxidation resistance ex vivo?

The ability of a range of dietary flavonoids to inhibit low-density lipoprotein (LDL) oxidation in vitro was tested using a number of different methods to assess oxidative damage to LDL. Overall quercetin was the most effective inhibitor of oxidative damage to LDL in vitro. On this basis, a diet enriched with onions and black tea was selected for a dietary intervention study that compared the effect on the Cu2+ ion-stimulated lag-time of LDL oxidation ex vivo in healthy human subjects of a high flavonoid diet compared with a low flavonoid diet. No significant difference was found in the Cu2+ ion-stimulated lag-time of LDL oxidation ex vivo between the high flavonoid and low flavonoid dietary treatments (48 +/- 1.6 min compared to 49 +/- 2.1 min).

A survey of white line disease in Japanese racehorses.

A survey was carried out into white line disease in 1781 Thoroughbred racehorses kept in stables at the Japan Racing Association (JRA) Miho Training Center (MTC) September-October 1996. The survey was conducted while horses were being shod by farriers. The horses that still exhibited damaged white lines after regular trimming were diagnosed as having white line disease. The factors recorded were age, sex, number of diseased horses, number of diseased hooves, number of lesions by region over the bearing border of the hoof and the classified length of such lesions. The percentage of total diseased horses was 11.5% (204 animals), with incidence increasing significantly with age (P< or =0.01). Occurrence was independent of sex (P>0.05) was more frequent in the fore- than in the hindhoof and developed more frequently at the toe than at any other region of the forehoof-bearing border. Most lesions ranged from 20 to 30 mm in length.

Development of a cell line system susceptible to infection with vaccine strains of MDV.

Despite reliance on the need to continually prepare fresh cultures of chick embryo fibroblasts (CEFs) to make Marek's disease (MD) vaccines, MD vaccines are the most widely used vaccines in the poultry industry. Preparation of CEF's accounts for approximately 40% of the costs associated with producing MD vaccines. A significant reduction in MD vaccine production costs could be realized if a continuous cell lines were available for MD vaccine production. Recently, we reported development and characterization of a cell line system (OCL) that supports growth and replication of oncogenic serotype 1 Marek's disease virus (MDV). Here we report development of three cell line systems for production of MD vaccine. These cell lines support the growth and replication of attenuated serotype 1 MDV (CVI-OCL), serotype 2 MDV (SB1-OCL) and serotype 3 MDV (HVT-OCL). MDV is maintained in a stable state in the OCL cells and the infected cells can be continuously grown. The vaccines made from these cell lines are safe and protect White Leghorn chickens against challenge with very virulent serotype 1 MDV, similar to traditional vaccines made from CEF cells. These cell line systems can significantly reduce the costs associated with MD vaccine production. Furthermore, the increased stability of MDV and the potential for positive selection of recombinant MDV suggest that OCL will be ideal for production of more effective MDV vaccines using recombinant DNA technology.

The number of copies of an a-like region in the serotype-3 Marek's disease virus DNA genome is variable.

A 1.2-kilobase-pair DNA region (a-like) in the serotype-3 Marek's disease virus was directly repeated at the ends of the long (L) and short (S) components of the genome and repeated in the opposite orientation at the junction between the L and S components. Four variants from plaque-purified virus that varied in the number of copies of the a-like region were identified. The predominant variant contained one copy at both ends of the genome and at the L-S junction. Two other variants contained either one or two copies at the L end and one to three copies at the L-S junction. The fourth variant lacked the a-like region at the L end and the L-S junction. The fourth variant became the predominate species as the virus was serially passaged in cell culture.

The quantitation and distribution of splicing intermediates in HeLa cells and adenovirus RNAs.

The steady state level of splicing intermediates in HeLa cells and in adenovirus RNA made late in the infectious cycle has been measured by a branch point analysis. About one in ten poly A(+) nuclear RNAs contained a branch point, but only 1/3 as many adenovirus RNAs were branched. Fewer branches were found in the poly A(-) RNAs of the nucleus and of late adenovirus transcripts suggesting that excised lariat introns do not accumulate in vivo. Branched RNAs were found in the poly A(+) RNAs from a nuclear matrix fraction, but several experiments failed to show an enrichment in these splicing intermediates in this matrix fraction. Branches were found in all size classes of poly A(+) nuclear RNA and were not exclusively associated with either the 3' or 5' regions, but were randomly distributed within RNA molecules. These results as well as the base and sequence data on branch points (1,18) are consistent with the conclusion that branched poly A(+) RNAs are splicing intermediates.

Fetal development of the white line (Zona alba) of the equine hoof.

The fetal development of the white line (Zona alba) in the equine hoof is described. Its specific structure of lamellar and interlamellar horn, which in turn is composed of cap and terminal horn, is formed in the second half of the hoof's fetal development. In equine fetuses with a crown-rump length of less than 550 mm, the hoof capsule lacks a 'characteristic' white line since no borders between stratum medium, stratum internum and sole horn are discernible. In the hoof of an equine fetus with a crown-rump length of 550 mm, a narrow white line has taken shape. Its shallow lamellae are arranged like arcades. Between the horn lamellae lie the polyhedral cells of the interlamellar horn. Up until birth, the height of the horn lamellae and, therefore, the width of the white line increases significantly. In the white line of the hoof of newborn foals, the terminal horn contains horn tubules with a characteristic architecture.

Effect of a supplementary dietary evening primrose oil mixture on hoof growth, hoof growth rate and hoof lipid fractions in horses: a controlled and blinded trial.

The lipid chemistry of the normal equine hoof, together with the effect of oral supplementation with an evening primrose oil mixture (EPOM) on its growth, growth rate and lipid content was assessed in a controlled and blinded feeding trial at the Defence Animal Centre. Twelve horses were paired as closely as possible according to sex, age, weight, height and colour and then one from each pair was randomly allocated to treatment or control groups. The treatment group received 30 ml of oral EPOM/day, otherwise the nutrition and management regimes were the same for all horses. No significant differences (P > 0.05) were seen between treatment and control groups for hoof horn growth or growth rate. However, there was a significant difference (P < 0.05) in hoof horn growth within the treatment group only between weeks 4 and 8 after the start of supplementation. The stratum medium contained significantly higher amounts of cholesterol ester (P < 0.05), triglycerides (P < 0.001) and free fatty acids (P < 0.05) than the periople. The periople contained significantly higher levels of free cholesterol and phospholipid (P < 0.001) than the stratum medium of the hoof wall. There were no significant differences (P > 0.05) between treatment and control groups for any of the lipid fractions measured for the stratum medium from the clippings of the hoof wall. However, there were differences in perioplic lipid analysis with significant increases (P < 0.05) in cholesterol esters and partial glycerides and a significant reduction (P < 0.001) in free cholesterol in the treatment group following supplementation.

The capsid polypeptides of the yeast viruses.

The yeast virus is a double-stranded RNA virus with a large genomic dsRNA and one major viral capsid polypeptide. Most strains of yeast have a major and a minor species of the large genomic dsRNA present. The major species has previously been shown to encode a capsid polypeptide with a Mr of about 88,000. We show that the minor species also encodes its capsid polypeptide with a Mr of about 80,000. Unlike all the dsRNA viruses of procaryotes and higher eucaryotes, the yeast virus appears to have only one major polypeptide in its virions. There are some 60 molecules of this capsid polypeptide per particle, consistent with a simple icosohedron of T=1.

Selection of Marek's disease virus recombinants expressing the Escherichia coli gpt gene.

We developed a positive selection method for recovering Marek's disease virus (MDV) recombinants. The Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt), under the control of the major immediate-early promoter from cytomegalovirus, was inserted into the inverted repeats flanking the unique long (UL) region of a non-pathogenic serotype 2 MDV strain 281MI/1. In a second demonstration of the usefulness of the positive selection system, the gpt gene was inserted into the inverted repeats flanking the unique short (US) region of the turkey herpesvirus (HVT) strain FC126. The targeted insertion site in 281MI/1 was in a previously established nonessential site for virus replication. The targeted insertion site for FC126, at the junction of the UL and US regions, is a nonessential site for in vitro replication of herpes simplex virus. Recombinant viruses were easily selected by incubating the transfected cells in mycophenolic acid (MPA)-containing medium. Purification of recombinants resulted from a series of trypsinization and sonication steps combined with the culturing of virus in MPA-containing medium to inhibit wild-type virus replication. This simple technique for recovering MDV and HVT recombinants should increase the efficiency of identifying nonessential sites and gene function analysis by insertional mutagenesis.

Tubule density of the stratum medium of horse hoof.

The number of tubules/mm2 (tubule density) of horse hoof horn was quantified in samples taken from the left forefeet of 8 randomly selected slaughterhouse horses in order to establish the normal tubule density characteristics at the midline dead centre (MDC) for the stratum medium of horse hoof. In the past the measurement of tubule distribution within the hoof has lacked objectivity. The horse hoof tubule density results are compared to a recent objective study carried out on pony hoof. A similar 4 zone pattern of tubule density was observed, although the precise zonal boundaries and tubule density values differed to those found for pony hoof. There were significant differences in tubule density between zones. Comparison with pony hoof revealed significant tubule density differences in zones 1, 2 and 4; however, there was no significant difference in zone 3. The existence of a 4 zoned pattern of tubule density for horse hoof, as for pony hoof, has been confirmed.

Effect of supplementary dietary biotin on hoof growth and hoof growth rate in ponies: a controlled trial.

The effect of dietary biotin supplementation, at a dose rate of 0.12 mg/kg bwt, on growth and growth rate of the hooves of 8 match-paired poines was investigated in a controlled feeding trial. Treatment animals had a mean hoof growth at the midline dead centre of the hoof capsule of 35.34 mm after 5 months of biotin supplementation compared to control animals 30.69 mm (P < 0.05). Comparison of regression analysis also showed that biotin supplementation produced a significantly higher (P < 0.02) growth rate of hoof horn in this trial. Treatment animals had a 15% higher growth rate of hoof horn and 15% more hoof growth at the midline dead centre, after 5 months of biotin supplementation compared to control ponies. No differences were found between feet for growth of horn, but the older animals in the trial had significantly lower hoof growth (P < 0.05) than the remaining poines.

Finite element analysis of static loading in donkey hoof wall.

A finite element model of donkey hoof wall was constructed from measurements taken directly from the hoof capsule of the left forefoot. The model was created with a 2 mm mesh and consisted of 11,608 nodes. A linear elastic analysis was conducted assuming isotropic material properties in response to a 375 newton (N) load, to simulate static loading. The load was applied to the wall via 400 laminae in order to simulate the way in which the pedal bone is suspended within the donkey hoof capsule. Displacement, stress concentration, principal strain, and force distribution across the hoof wall were evaluated. The hoof wall model revealed loading responses that were in broad agreement with previously reported in vivo and modelled observations of the equid hoof. Finite element analysis offers the potential to model hoof wall function at the macroscopic and microscopic level. In this way, it could help to develop further our understanding of the functional relationship between the structural organisation and material properties of the hoof wall.