A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: Total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH2S)(28-29,56-57,76-77)]

The 101 residue protein early pregnancy factor (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH2CH2SH thiolate with XCH2CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH2CH2SH thiolate with a BrCH2CO-peptide than with a CICH2CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N-alpha-chloroacetyl, C-terminal thiolated peptide with a second N-alpha-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N-alpha-chloroacetyl peptides could be activated by halide exchange using saturated KI solutions to yield the highly reactive No-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or cassettes comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH2CH2SH] 13, ClAc-[EPF 30-55]-[NHCH2CH2SH] 14, and Ac-[EPF 1-27]-[NHCH2CH2SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH2S)(28-29,56-57,76-77)) 19 in 19% overall yield.

The efficacy of a vitamin D-3 metabolite for improving the myofibrillar tenderness of meat from Bos indicus cattle

The influence of a once only administration of a metabolite of vitamin D-3 (HY center dot D-(R)-25-hydroxy vitamin D-3) on myofibrillar meat tenderness in Australian Brahman cattle was studied. Ninety-six Brahman steers of three phenotypes (indo-Brazil, US and US/European) and with two previous hormonal growth promotant (HGP) histories (implanted or not implanted with Compudose((R))) were fed a standard feedlot ration for 70 d. Treatment groups of 24 steers were offered daily 10 g/head HY center dot D-(R) (125 mg 25-hydroxyvitamin D-3) for 6, 4, or 2 d before slaughter. One other group of 24 steers was given the basal diet without HY center dot D-(R). Feed lot performance, blood and muscle samples and carcass quality data were collected at slaughter. Calcium, magnesium, potassium, sodium, iron and Vitamin D-3 metabolites were measured in plasma and longissimus dorsi muscle. Warner-Bratzler (WB) shear force (peak force, initial yield) and other objective meat quality measurements were made on the longissimus dorsi muscle of each steer after ageing for 1, 7 and 14 d post-mortem at 0-2 degrees C. There were no significant effects of HY center dot D-(R) supplements on average daily gain (ADG, 1.28-1.45 kg/d) over the experimental period. HY center dot D-(R) supplements given 6 d prior to slaughter resulted in significantly higher (P < 0.05) initial yield values compared to supplements given 2 d prior to slaughter. Supplementation had no significant effect on meat colour, ultimate pH, sarcomere length, cooking loss, instron compression or peak force. There was a significant treatment (HY center dot D-(R)) by phenotype/HGP interaction for peak force (P = 0.028), in which Indo-Brazil steers without previous HGP treatment responded positively (increased tenderness) to HY center dot D-(R) supplements at 2 d when compared with Indo-Brazil steers previously given HGP. There were no significant effects of treatment on other phenotypes. HY center dot D-(R) supplements did not affect muscle or plasma concentrations of calcium, potassium or sodium, but did significantly decrease plasma magnesium and iron concentrations when given 2 d before slaughter. There were no detectable amounts of 25-hydroxyvitamin D-3 in the blood or muscle of any cattle at slaughter. (c) 2005 Elsevier Ltd. All rights reserved.

The effect of temperature on the size and population density of dinoflagellates in larvae of the reef coral Porites astreoides

Pre-settlement events play an important role in determining larval success in marine invertebrates with bentho-pelagic life histories, yet the consequences of these events typically are not well understood. The purpose of this study was to examine the pre-settlement impacts of different seawater temperatures on the size and population density of dinoflagellate symbionts in brooded larvae of the Caribbean coral Porites astreoides. Larvae were collected from P. astreoides at 14-20 m depth on Conch Reef (Florida) in June 2002, and incubated for 24 h at 15 temperatures spanning the range 25.1 degrees-30.0 degrees C in mean increments of 0.4 +/- 0.1 degrees C (+/- SD). The most striking feature of the larval responses was the magnitude of change in both parameters across this 5 degrees C temperature range within 24 h. In general, larvae were largest and had the highest population densities of Symbiodinium sp. between 26.4 degrees-27.7 degrees C, and were smallest and had the lowest population densities at 25.8 degrees C and 28.8 degrees C. Larval size and symbiont population density were elevated slightly (relative to the minimal values) at the temperature extremes of 25.1 degrees C and 30 degrees C. These data demonstrate that coral larvae are highly sensitive to seawater temperature during their pelagic phase, and respond through changes in size and the population densities of Symbiodinium sp. to ecologically relevant temperature signals within 24 h. The extent to which these changes are biologically meaningful will depend on the duration and frequency of exposure of coral larvae to spatio-temporal variability in seawater temperature, and whether the responses have cascading effects on larval success and their entry to the post-settlement and recruitment phase.

The pearl oysters, Pinctada maxima and P margaritifera, respond in different ways to culture in dissimilar environments

Growth, Condition Index (CI) and survival of the pearl oysters, Pinctada maxima and R margaritifera, were measured in three size groups of oysters over 14 months at two dissimilar environments in the Great Barrier Reef lagoon. These were the Australian Institute of Marine Science (AIMS) in a mainland bay and Orpheus Island Research Station (OIRS) in coral reef waters. Temperature, suspended particulate matter (SPM) and particulate organic matter (POM) were monitored during the study. Temperature at AIMS fluctuated more widely than at OIRS both daily and seasonally, with annual ranges 20-31 degrees C and 22-30 degrees C, respectively. Mean SPM concentration at AIMS (11.1 mg l(-1)) was much higher than at OIRS (1.4 mg l(-1)) and fluctuated widely (2-60 mg l(-1)). Mean POM level was also substantially higher at AIMS, being 2.1 mg l(-1) compared with 0.56 mg l(-1) at OIRS. Von Bertalatiffy growth curve analyses showed that P. maxima grew more rapidly and to larger sizes than P. margaritifera at both sites. For the shell height (SH) of R maxima, growth index phi'=4.31 and 4.24, asymptotic size SHinfinity = 229 and 205 mm, and time to reach 120 mm SH (T-(120))= 1.9 and 2.1 years at AIMS and OIRS, respectively. While for P margaritifera, phi'=4.00 and 4.15, SHinfinity = 136 and 157 mm, and T-(120) = 2.5 and 3.9 years at AIMS and OIRS, respectively. R maxima had significantly lower growth rates and lower survival of small oysters during winter compared with summer. There were, however, no significant differences between the two sites in growth rates of P. maxima and final Cl values. In contrast, P. margaritifiera showed significant differences between sites and not seasons, with lower growth rates, survival of small oysters, final Cl values and asymptotic sizes at AIMS. The winter low temperatures, but not high SPM at AIMS, adversely affected P. maxima. Conversely, the high SPM levels at AIMS, but not temperature, adversely affected P. margaritifera. This was in accordance with earlier laboratory-based energetics studies of the effects of temperature and SPM on these two species. P maxima has potential to be commercially cultured in ca. > 25 degrees C waters with a wide range of SPM levels, including oligotrophic coral reef waters with appropriate particle sizes. It is possible to culture R margaritifera in turbid conditions, but its poor performance in these conditions makes commercial culture unlikely. (c) 2005 Elsevier B.V. All rights reserved.

Synchronization of estrus and ovulation and associated endocrine changes in Bos indicus cows

The effects of 4 estrus synchronization treatments on intervals to and synchrony of estrus and ovulation, on timing of the preovulatory LH surge and associated changes in plasma progesterone, LH, FSH, and 17 beta-estradiol (E(2)) were investigated in 48 Bos indicus cows. Treatment 1 consisted of 2 injections of PGF(2 alpha) 14 d apart (n = 12); Treatment 2 of a subcutaneous 3-mg norgestomet implant and an intramuscular injection of 3 mg of norgestomet and 5 mg estradiol valerate, with the implant removed 10 d later (n = 12; norgestomet-estradiol); Treatment 3 of norgestomet-estradiol, with a subcutaneous injection of PMSG given at time of implant removal (Day 10; n = 12); and Treatment 4 of norgestomet implant (as for Treatments 2 and 3) inserted for 10 d, with an intramuscular injection of PGF(2 alpha) given at the time of implant removal (n = 12). The experiment was conducted in 2 replicates (24 cows/replicate, 6 cows/group). Estrus, ovulation and timing of the preovulatory surge of LH varied less in cows treated with norgestomet-estradiol and PMSG than in cows in Treatments 1 and 4 (P < 0.008). Treatment with PMSG;educed variation in ovulation times and timing of the LH surge in cows treated with norgestomet-estradiol (P < 0.02). Concentrations of E(2) were higher in cows in Treatments 2 and 3 on the final day of treatment and at about 6 h post ovulation compared with cows in Treatments 1 and 4 (P < 0.05). Different methods for synchronizing estrus did not alter sequential endocrine and behavioral changes in relation to the timing of the LH peak, and the results were consistent with current recommendations for insemination times in Bos taurus cattle. (C) 1997 by Elsevier Science Inc.

Pulmonary Vascular remodelling in hypoxic rats: effects of amlodipine, alone and with perindopril

This study investigated whether pulmonary Vascular remodelling in hypoxic pulmonary hypertensive rats (10% oxygen; 4 weeks) could be prevented by treatment, during hypoxia, with amlodipine (IO mg/kg/day, p.o.), either alone or in combination with the angiotensin converting enzyme inhibitor, perindopril (30 mg/kg/day, p.o.). Medial thickening of pulmonary arteries (30-500 mum o.d.) was attenuated by amlodipine whereas it was totally prevented by the combination treatment (amlodipine plus perindopril); neomuscularisation of small alveolar arteries (assessed from critical closing pressure in isolated perfused lungs) was not affected. Pulmonary vascular resistance (isolated perfused lungs) was reduced by both treatment regimes but only combination treatment reduced right ventricular hypertrophy. Taus, amlodipine has anti-remodelling properties in pulmonary hypertensive rats. The finding that combining amlodipine with another anti-remodelling drug produced effects on vascular structure that were additive raises the question of whether combination therapy with two different anti-remodelling drugs may be of value in the treatment of patients with hypoxic (and possibly other forms of) pulmonary hypertension. (C) 2001 Elsevier Science B.V. All rights reserved.

Comparison of tafenoquine (WR238605) and primaquine in the post-exposure (terminal) prophylaxis of vivax malaria) in Australian Defence Force personnel

On return from duty in North Solomons Province (including Bougainville Island), Papua New Guinea, 586 Australian Defence Force personnel received either primaquine (14-d) or tafenoquine (3-d) post-exposure malaria prophylaxis. Within 12 months, 6 of the 214 volunteers receiving primaquine and 7 of 378 receiving tafenoquine had developed vivax malaria. Overall, volunteers preferred the shorter course of tafenoquine.

Enhancement or modulation of the vector competence of ochlerotatus vigilax (Diptera : Culicidae) for Ross River virus by temperature

Two different doses of Ross River virus (1111) were fed to Ochlerotatus vigilax (Skuse), the primary coastal vector in Australia; and blood engorged females were held at different temperatures up to 35 d. After ingesting 10(4.3) CCID50/Mosquito, mosquitoes reared at 18 and 25degreesC (and held at the same temperature) had higher body remnant and head and salivary gland titers than those held at 32degreesC, although infection rates were comparable. At 18, 25, and 32degreesC, respectively, virus was first detected in the salivary glands on days 3, 2, and 3. Based on a previously demonstrated 98.7% concordance between salivary gland infection and transmission, the extrinsic incubation periods were estimated as 5, 4, and 3 d, respectively, for these three temperatures. When Oc. vigilax reared at 18, 25, or 32degreesC were fed a lower dosage of 10(3.3) CCID50 RR/mosquito, and assayed after 7 d extrinsic incubation at these (or combinations of these) temperatures, infection rates and titers were similar. However, by 14 d, infection rates and titers of those reared and held at 18 and 32degreesC were significantly higher and lower, respectively. However, this process was reversible when the moderate 25degreesC was involved, and intermediate infection rates and titers resulted. These data indicate that for the strains of RR and Oc. vigilax used, rearing temperature is unimportant to vector competence in the field, and that ambient temperature variations will modulate or enhance detectable infection rates only after 7 d: extrinsic incubation. Because of the short duration of extrinsic incubation, however, this will do little to influence RR epidemiology, because by this time some Oc. vigilax could be seeking their third blood meal, the latter two being infectious.

Field evaluation of a sentinel mosquito (Diptera : culicidae) trap system to detect Japanese encephalitis in remote Australia

Incursions of Japanese encephalitis (JE) virus into northern Queensland are currently monitored using sentinel pigs. However, the maintenance of these pigs is expensive, and because pigs are the major amplifying hosts of the virus, they may contribute to JE transmission. Therefore, we evaluated a mosquito-based detection system to potentially replace the sentinel pigs. Single, inactivated JE-infected Culex annulirostris Skuse and C. sitiens Wiedemann were placed into pools of uninfected mosquitoes that were housed in a Mosquito Magnet Pro (MM) trap set under wet season field conditions in Cairns, Queensland for 0, 7, or 14 d. JE viral RNA was detected (cycling threshold [CT] = 40) in 11/ 12, 10/14, and 2/5 pools containing 200, 1,000, and 5,000 mosquitoes, respectively, using a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability to detect virus was not affected by the length of time pools were maintained under field conditions, although the CT score tended to increase with field exposure time. Furthermore, JE viral RNA was detected in three pools of 1,000 mosquitoes collected from Badu Island using a MM trap. These results indicated that a mosquito trap system employing self-powered traps, such as the MosquitoMagnet, and a real-time PCR system, could be used to monitor for JE in remote areas.

Secular trend in the development of fatness during childhood and adolescence

This study examined the development of fatness, as indexed by skinfold thickness, in healthy Caucasian children and adolescents residing in the same location in Canada in the 1960s and the 1990s. The data comes from two longitudinal studies, conducted approximately 30 years apart, of children aged 8-16 years. The first study (1964-1973) annually measured 207 males and 140 females. The second investigation (1991-1997) repeatedly measured 113 males and 115 females. Identical measurement tools and protocols were used for height, body mass, and skinfolds. Maturational age was estimated as a measure in years from age of peak height velocity. Males from the second investigation matured significantly (P < 0.05) earlier. Multilevel regression modeling was utilized to determine developmental curves for the individuals within the two populations. When differences in height, body mass, and maturity were controlled, skinfold thicknesses of the males and females in the second study were significantly greater (P < 0.05) than age- and sex-matched peers in the first study. This was not seen in models of the BMI. The results suggest that when maturity and size were controlled, the fatness of children and adolescents increased over 30 years. (C) 2002 Wiley-Liss, Inc.

Testing the 'photoinhibition' model of coral bleaching using chemical inhibitors

Explants of the hard coral Seriatopora hystrix were exposed to sublethal concentrations of the herbicide diuron DCMU (N'-(3,4-dichlorophenyl,-N,N-dimethylurea)) and the heavy metal copper. Pulse amplitude modulated (PAM) chlorophyll fluorescence techniques were used to assess the effects on the photosynthetic efficiency of the algal symbionts in the tissue (in Symbio), and chlorophyll fluorescence and counts of symbiotic algae (normalised to surface area) were used to assess the extent of coral bleaching. At 30 mug DCMU l(-1), there was a reduction in both the maximum effective quantum yield (DeltaF/F-m') and maximum potential quantum yield (F-v/F-m) of the algal symbionts in symbio. Corals subsequently lost their algal symbionts and discoloured (bleached), especially on their upper sunlight-exposed surfaces. At the same DCMU concentration but under low light (5% of growth irradiance), there was a marked reduction in DeltaF/F-m' but only a slight reduction in F-v/F-m and slight loss of algae. Loss of algal symbionts was also noted after a 7 d exposure to concentrations as low as 10 mug DCMU l(-1) under normal growth irradiance, and after 14 d exposure to 10 mug DCMU l(-1) under reduced irradiance. Collectively the results indicate that DCMU-induced bleaching is caused by a light-dependent photoinactivation of algal symbionts, and that bleaching occurs when F-v/F-n, (measured 2 h after sunset) is reduced to a value of less than or equal to 0.6. Elevated copper concentrations (60 mug Cu l(-1) for 10 h) also induced a rapid bleaching in S. hystrix but without affecting the quantum yield of the algae in symbio. Tests with isolated algae indicated that substantially higher concentrations (300 mug Cu l(-1) for 8 h) were needed to significantly reduce the quantum yield. Thus, copper-induced bleaching occurs without affecting the algal photosynthesis and may be related to effects on the host (animal). It is argued that warm-water bleaching of corals resembles both types of chemically induced bleaching, suggesting the need for an integrated model of coral bleaching involving the effect of temperature on both host (coral) and algal symbionts.

Increased calcium influx mediates increased cardiac stiffness in hyperthyroid rats

Cardiac remodeling (hypertrophy and fibrosis) and an increased left ventricular diastolic stiffness characterize models of hypertension such as the SHR and DOCA-salt hypertensive rats. By contrast, hyperthyroidism induces hypertrophy and hypertension, yet collagen expression and deposition is unchanged or decreased, whereas diastolic stiffness is increased. We determined the possible role of increased calcium influx in the development of increased diastolic stiffness in hyperthyroidism by administering verapamil (15 mg/[kg(.)d] orally) to rats given triiodothyronine (T-3) (0.5 mg/[kg.d] subcutaneously for 14 d). Administration of T3 significantly increased body temperature (control: 36.7 +/- 0.2 degrees C; T-3: 39.6 +/- 0.2 degrees C), left ventricular wet weight (control: 2.09 +/- 0.02 mg/kg; T-3 3.07 +/- 0.07 mg/kg), systolic blood pressure (control: 128 +/- 5 mmHg; T-3: 156 +/- 4 mmHg), and left ventricular diastolic stiffness (control: 20.6 +/- 2.0; T-3: 28.8 +/- 1.4). Collagen content of the left ventricle was unchanged. Contractile response to noradrenaline in thoracic aortic rings was reduced. Relaxation in response to acetylcholine (ACh) was also reduced in T-3-treated rats, whereas sodium nitroprusside response was unchanged. Verapamil treatment of hyperthyroid rats completely prevented the increased diastolic stiffness and systolic blood pressure while attenuating the increased body temperature and left ventricular weight; collagen content remained unchanged. ACh response in thoracic aortic rings was restored by verapamil. Thus, in hyperthyroid rats, an increased calcium influx is a potential mediator of the increased diastolic stiffness independent of changes in collagen.

Effect of harvest time and drying on supersweet sweet corn seed quality

A supersweet sweet corn hybrid, Pacific H5, was grown under field conditions in South-East Queensland to study the effects of harvest time and drying conditions on seed quality. Cobs were harvested at different times to obtain seed with two moisture percentage ranges (20-30% and 40-50%) and dried to 12% moisture under different combinations of drying temperatures (30 degrees C, 40 degrees C and 50 degrees C) and air velocities (1.25 m/s, 2.75 m/s and 4.30 m/s). Dried seed was stored at 30 degrees C with bimonthly monitoring of seed quality for 12 months. For standard as well as cold test germinations, statistical analysis yielded significant main effects for temperature, air velocity and harvest moisture content and significant interactions for drying temperature by harvest moisture and drying temperature by air velocity. Germination at the beginning of storage was unaffected by drying temperatures up to 40 degrees C regardless of harvest moisture but was lower at 50 degrees C for higher moisture. However, germination at the end of the storage period of 12 months was greatest for seed harvested at higher moisture and dried at temperatures up to 40 degrees C. Germination was not affected by air velocity for drying temperatures up to 40 degrees C but at 50 degrees C it generally decreased with increase in air velocity. To slow down seed deterioration during storage, it is recommended that sweet corn seed should be harvested at a higher moisture range (40-50%) and dried at 40 degrees C and 4.30 m/s air velocity. The drying temperature can be raised to 50 degrees C for seed harvested at a low moisture range (20-30%) provided the air velocity is kept low (1.25 m/s).

Effect of modified atmosphere packaging on the quality of minimally processed pineapple cv.'Smooth Cayenne' fruit

The effects of modified atmosphere (MA) conditions on the quality of minimally processed pineapple slices were determined. Commercial pineapple slice packs sealed with 40 pm thick polyester film were kept at 4.5 degrees C for 14 d. The oxygen transmission rate of the film was 23 ml m(-2) day(-1) atm(-1) (at 25 degrees C, 75% RH). In-built atmospheres and the quality of the products were determined. O-2 concentrations within the packs stabilised at 2%, while CO2 concentrations increased to 70% by day 14. The high CO2 level suggested an inappropriate lidding film permeability for the product, and hence affected its quality. Three batches of pineapple slices were packed in the laboratory using lidding films with oxygen transmission rate of 75, 2790 or 5000 ml m(-2) day(-1) atm(-1) (at 23 degrees C, 0% RH). Headspace atmospheres from laboratory-packed pineapple slices suggested an optimum equilibrium modified atmosphere of ca. 2% O-2 and 15% CO2. Respiration data from the laboratory-prepared packs were pooled together and used to develop a correlation model relating respiration rates to O-2 and CO2 concentrations. The model showed a decrease in respiration rate with decreasing O-2 and increasing CO2 concentrations. Respiration rate stabilised at 2% 02 and 10% CO2. The high concentrations of CO2 observed in the commercial packs did not fit the range in the respiration model. The model could aid in selection of MA conditions for minimally processed pineapple fruit.