Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants

A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.     

A common allele on chromosome 9 associated with coronary heart disease

Coronary heart disease (CHD) is a major cause of death in Western countries. We used genome-wide association scanning to identify a 58-kilobase interval on chromosome 9p21 that was consistently associated with CHD in six independent samples (more than 23,000 participants) from four Caucasian populations. This interval, which is located near the CDKN2A and CDKN2B genes, contains no annotated genes and is not associated with established CHD risk factors such as plasma lipoproteins, hypertension, or diabetes. Homozygotes for the risk allele make up 20 to 25% of Caucasians and have a approximately 30 to 40% increased risk of CHD.     

Vitrification with Glutamine Improves Maturation Rate of Vitrified / Warmed Immature Bovine Oocytes

The current study examined the protective effects of l ‐glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 μg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. l ‐glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).     

Absence of a Grass/Fire Cycle in a Semiarid Grassland: Response to Prescribed Fire and Grazing

Many nonnative invasive grasses alter fire regimes to their own benefit and the detriment of native organisms. In southern Arizona the nonnative Lehmann lovegrass (Eragrostis lehmanniana Nees) dominates many semiarid grasslands where native grasses were abundant. Managers are wary of using prescribed fire in this fire-prone community partly due to the perceived effects of a grass/fire cycle. However, examples of the grass/fire cycle originate in ecosystems where native plants are less fire-tolerant than grasses and the invasive plant does not mimic the physiognomy of the native community. We investigate the effects of prescribed fire and livestock grazing on a semiarid grassland community dominated by a nonnative invasive grass. Lehmann lovegrass does not appear to alter the fire regime of semiarid grasslands to the detriment of native plants. Prescribed fire reduced the abundance of Lehmann lovegrass for 1 to 2 yr while increasing abundance of native grasses, herbaceous dicotyledons and fall richness, and diversity. Effects of livestock grazing were less transformative than the effects of fire in this long-grazed area, but grazing negatively affected native plants as did the combination of prescribed fire and livestock grazing. Although Lehmann lovegrass produces more fuel than native plants, fire frequency in semiarid grasslands appears to be limited by the paucity of above-average precipitation, which constrains high fuel loads. In addition, many native grasses tolerate high temperatures produced by Lehmann lovegrass fires. Consistent with previous research, fire does not promote the spread of Lehmann lovegrass, and more importantly human alteration of the fire regime is greater than the nominal effects of Lehmann lovegrass introduction on the fire regime.     

Assessing East African trade in seahorse species as a basis for conservation under international controls

• 1. Seahorses (Hippocampus spp.), many of which are listed as Vulnerable or Endangered on the IUCN Red List, are traded worldwide as souvenirs, aquarium fish and, primarily, for use in traditional medicines. Given concern over the sustainability of this trade, the genus was added to Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in May 2004.
• 2. This paper reports findings of the first ever survey of seahorse trade in Africa, conducted in Kenya and Tanzania in May and June 2000.
• 3. Seahorse trade in Kenya was found to be negligible, with approximately 10 live seahorses exported as aquarium fish annually. Until 1998, however, Kenya may have imported somewhere from 1 to 2.3 t of dried seahorses annually from Tanzania for re‐export to Asian medicine markets. Seahorse trade in Tanzania remained substantial, with at least 630–930 kg of dried seahorse exported directly to Asia each year.
• 4. Accounts of declines in seahorse availability and seahorse size, although few in number, could be early warning signs that wild populations are suffering, at least locally. Close monitoring of future developments in the trade will be essential to allow for timely conservation action as and when necessary, and would contribute to our understanding of the ecological and economical implications of small‐scale, non‐food fisheries.

Copyright © 2004 John Wiley & Sons, Ltd.     

Responses of oaks and tanoaks to the sudden oak death pathogen after 8 y of monitoring in two coastal California forests

Sudden oak death, caused by Phytophthora ramorum, is widely established in mesic forests of coastal central and northern California. In 2000, we placed 18 plots in two Marin County sites to monitor disease progression in coast live oaks (Quercus agrifolia), California black oaks (Q. kelloggii), and tanoaks (Lithocarpus densiflorus), the species that are most consistently killed by the pathogen in these areas. Through early 2008, the numbers of newly infected trees increased for all species. The infection rate for trees that were asymptomatic in 2000 was 5.0% y⁻¹ for coast live oaks, 4.1% y⁻¹ for black oaks and 10.0% y⁻¹ for tanoaks. Mortality rates were 3.1% y⁻¹ for coast live oaks, 2.4% y⁻¹ for black oaks, and 5.4% y⁻¹ for tanoaks. Mortality not attributed to P. ramorum was 0.54% y⁻¹ for coast live oaks, and 0.75% y⁻¹ for tanoaks. Weibull survival models of trees that were asymptomatic in 2000 provided overall median survival times of 13.7 y for coast live oaks, 13.8 y for black oaks, and 8.8 y for tanoaks. Survival of infected (bleeding) trees declined to 9.7 y for coast live oaks, 6.2 y for black oaks, and 5.8 y for tanoaks. Ambrosia beetle attacks on bleeding trees further reduced modeled survival times by 65–80%, reaffirming the earlier finding that beetle attacks on bleeding cankers considerably reduce survival. Across all plots, the modeled time for 90% of trees that were asymptomatic in 2000 to become infected is 36.5 y for coast live oaks and 15.4 y for tanoaks. There was a trend toward higher infection rates as tree diameter increased. Greater than 90% of living coast live oaks that failed during the study had extensive beetle tunneling at the site of the break. Disease intensity in coast live oaks at the plot level was positively associated with bay laurel (Umbellularia californica) basal area and negatively associated with Pacific madrone (Arbutus menziesii) basal area. This study demonstrates the use of survival modeling to characterize the effects of epidemic disease on different species and to project the future of forests infected with tree pathogens.     

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia

Ninety‐three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.     

A study on the possible role of chymotrypsin in the aetiology of equine chronic obstructive pulmonary disease (COPD)

The chymotrypsin activity of seven batches of Micropolyspora faeni and of five batches of Aspergillus fumigatus culture extracts, prepared for inhalation challenge in horses, was assayed and was found to range between 0.29 and 1.45 units/mg protein and 0.02 and 0.20 units/mg protein respectively. Horses affected with chronic obstructive pulmonary disease (COPD) were challenged with two batches of each antigen which had different chymotrypsin activities and no significant correlations were found between the degree of response to challenge and the chymotrypsin activity of the antigens. Inhalation of two doses of nebulised, purified chymotrypsin over 4 days did not induce signs of respiratory disease in COPD-affected horses. However, repeated chymotrypsin inhalations after an interval of 3 weeks caused an exacerbation of signs of COPD in one horse. These studies suggest that, although repeated inhalation of purified chymotrypsin may induce respiratory hypersensitivity in horses, the chymotrypsin-like enzymes of M. faeni and A. fumigatus do not play a major role in the precipitation of clinical signs of equine COPD.