Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities

The binding and assembly of clathrin triskelions on vesicle membranes seem to be mediated by certain assembly polypeptides (Keen, J.H., Willingham, M.C., and Pastau, I.H. (1979) Cell 16, 303-312). These assembly polypeptides were further purified into two distinct complexes using hydroxylapatite chromatography. Peak 1 consists of two major bands of 98 and 112 kDa, two minor bands of 103 and 118 kDa, and a polypeptide of 46 kDa. Peak 2 consists of one major band of 100 kDa, two minor bands of 103 and 115 kDa, and a polypeptide of 50 kDa. Both complexes have a native molecular mass of 290 kDa as determined by gel filtration. Each 290-kDa complex contains two polypeptides of 98-118/100-115 kDa and two polypeptides of 46/50 kDa. The 46-kDa polypeptide is not phosphorylated, whereas the 50-kDa polypeptide is. Both peaks contain 50-kDa kinase-like activity. Time courses of the 50-kDa phosphorylation show that the activity in peak 1 saturates much faster than the activity in peak 2; there may be two 50-kDa kinase activities in coated vesicles. A kinase that phosphorylates the polypeptides in 98-118-kDa group is present in peak 1 but not in peak 2. Both peaks assemble clathrin triskelions into cages under conditions in which the clathrin alone would not assemble. Both rotary shadowed and negatively stained preparations of these reassembled cages as well as the purified complexes were examined by electron microscopy. Thus, two complexes have been identified that differ in their polypeptide composition and kinase activities, but are similar in their ability to assemble clathrin triskelions into cages.     

Housing mice in a caging system with automatic flushing.

Male Har:(ICR)BR mice were housed 8 wk in wire bottom cages over paper, in wire bottom cages over an automatic cascade flushing system, or in solid-bottom plastic cages with bedding material. There were no substantial differences in general health or weight gain. Pentobarbital LD50 values were lower for the mice housed in wire bottom cages than for the mice in solid bottom cages with bedding. This difference was probably related to gastrointestinal content. It appears that the automatic cascade flushing system is suitable for housing mice for periods up to 8 wk.     

Calmodulin binding and protein phosphorylation in adrenal medulla coated vesicles

Coated vesicles from bovine adrenal medulla contained clathrin and major detergent-insoluble polypeptides of 120-100, 51 and 49 kDa. Intact coated vesicles and vesicles lacking clathrin light chains were bound by immobilized calmodulin in the presence of Ca2+. Clathrin in the form of 700 A cages was not bound. The calmodulin binding components in intact coated vesicles are therefore contributed by the enclosed vesicle or by the 120-100, 50 or 49 kDa polypeptides. The 51 kDa component incorporated 32Pi from labelled ATP by an endogenous kinase activity; no other coat or vesicle membrane protein was phosphorylated in vitro, either by intrinsic or exogenous kinases.     

Field-Cage Studies of Beauveria bassiana (Hyphomycetes: Moniliaceae) for the Suppression of Adult Western Corn Rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

The efficacy of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin, was tested as a control agent for adult western corn rootworm, Diabrotica virgifera virgifera LeConte, in walk-in field cages. Suspensions of B. bassiana conidia were applied to corn plants in cages into which laboratory-reared beetles had been released. Beetles were collected at 3 and 5 days post-application and evaluated in the laboratory for mortality. Mortality was 10, 29 and 50%, at rate equivalents of 7 X 10 12 , 2 X 10 13 (two applications), and 5 X 10 13 conidia/ ha, respectively. There was no significant difference in mortality of beetles collected at 3 days compared with 5 days post-application. Mortality due to B. bassiana was 24% when beetles were released into field cages 24 h post-application (5 X 10 13 conidia/ha) compared with 50% when beetles were present during the application. Beetle mortality declined significantly with increasing time from application in feeding assays carried out with leaf samples removed from plants at 0, 12, 24 and 72 h post-application. Mortality of beetles collected from treated plants within cages and maintained in the laboratory was found to overestimate the population decline by 10% when compared with beetle estimates from treated plants within field cages.     

Laboratory culture conditions affect stability of resistance to Bacillus thuringiensis Cry1Ac in Plutella xylostella (Lep., Plutellidae)

Abstract:  Populations of Plutella xylostella L. with high (laboratory re-selected) and low levels (unselected) of resistance to the Bacillus thuringiensis ( B.t. ) toxin Cry1Ac were maintained under controlled environment conditions in the absence of any further selection pressure. Larvae were maintained either in discrete generations on freshly excised leaves replaced ad lib (standard culture system) or in microcosm cages holding up to 12 potted Chinese cabbage plants, allowing progressively overlapping generations of insects. After 12 generations in the microcosm cages, the LC₅₀ values of both selected and unselected populations were not significantly different from those of a standard susceptible population. Under standard culture conditions, the LC₅₀ value for unselected insects after 12 generations was almost identical to that of unselected insects in the microcosm cages, whereas the LC₅₀ value for selected insects was 1250-fold greater. The fecundity of the selected population was significantly lower compared with that of the same population in the microcosm cages after 12 generations. There was no significant change in the fecundity of the unselected population after 12 generations in the microcosm cages. The results show, that differences in the culture system (microcosm vs. standard) and in the resistance level (re-selected vs. unselected) can have a marked influence on the stability of resistance to a B.t. toxin in an insect population. Possible factors involved in determining the rate at which resistance declines are discussed.     

Effect of acclimation to caging on nephrotoxic response of rats to uranium

Animal studies of the toxicity and metabolism of radionuclides and chemicals often require housing of rats in metabolism cages for excreta collection. Response of rats to toxic substances may be affected by environmental factors such as the type of cage used. Dose-response studies were conducted to assess the effects of two types of cages on the nephrotoxic response of rats to uranium from implanted refined uranium ore (yellowcake). The LD50/21 days was 6 mg of uranium ore per kilogram body weight (6 mg U/kg). The 95% confidence limit (C.L.) was 3-8 mg U/kg for rats housed in metabolism cages beginning on the day of implantation (naive rats). However, for rats housed in metabolism cages for 21 days before implantation (acclimated rats) the LD50/21 days was 360 mg U/kg (95% C.L. = 220-650 mg U/kg), which was the same value obtained for rats housed continuously in polycarbonate cages. This significant difference (P less than 0.01) in response of naive rats compared to response of acclimated rats appeared related to a significantly lower water consumption by the naive rats.     

Preferences of farmed blue foxes for different floor types

In the first experiment, farmed blue foxes were allowed to choose for 1 week between four standard farm cages equipped with different floor materials: plastic-coated wire mesh, dry wood, dry sand and wet (summer) or icy (winter) sand. Resting consisted of 10-15 separate bouts/day occupying 50-60% of the total 24-h. There were no other differences in the use of the cages except that the time spent on, and the duration and number of resting bouts were lower on wet or icy sand, resting periods being more affected than activity. In the second experiment, two cages were connected with a 1.2 m tunnel. One cage was always elevated (50 cm) compared to that one which was lower. One cage of each pair had a wire floor whereas the other cage either had sand or wire floor. Sand floor was preferred for active behaviours and wire floor for resting if these were on elevated level. Of two identical wire-floored cages, the elevated one had the priority. Foxes preferred to rest on the same floor where they had finished their previous resting bout, often exclusively and independently of floor material or floor level.     

Reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus

Serologic monitoring of sentinel mice exposed to soiled bedding is a common method of detecting viral infections in mice. Because bedding transfer protocols vary, the sensitivity of this method has not been documented sufficiently. We examined the reliability of bedding transfer during various stages of infection with mouse parvovirus (MPV) and mouse hepatitis virus (MHV). Most mice exposed to bedding contaminated with MPV 0, 3, or 7 d previously seroconverted, whereas only mice exposed to bedding contaminated with MHV 4 h previously seroconverted, thus confirming the differing stabilities of these viruses. Index mice were inoculated with 30 times the infectious dose 50 (ID50) of MPV or 300 ID50 of MHV. At 3 d, 1 wk, and 2 wk postinoculation (PI), we transferred 25, 50, or 100 ml of bedding to cages of sentinel mice. Viral infection and shedding by index mice was confirmed by serology and fecal polymerase chain reaction assay. Transfer of soiled bedding between mice in static cages induced seroconversion of sentinel mice most reliably during peak viral shedding (1 wk PI for MPV and 3 d PI for MHV). Soiled bedding transfer between mice in individually ventilated cages induced a higher prevalence of sentinel seroconversion to MPV and MHV than that after transfer between mice in static cages. Our findings indicate that although soiled bedding transfer is an effective method for detecting MHV and MPV under optimal conditions, the method is less than 100% reliable under many conditions in contemporary mouse facilities.     

Influence of housing conditions on pregnancy outcome in cynomolgus monkeys (Macaca fascicularis)

Female cynomolgus monkeys (Macaca fascicularis) were kept under 3 different housing conditions: individually in type A cages (45 X 45 X 60 cm), individually in type B cages (70 X 70 X 100 cm) and as couples in type B cages. Primigravida did not show early embryonic mortality, differing significantly from 11.5% early losses in multigravida. Early embryonic mortality was not affected by housing condition. Further reproductive failure rates did not differ significantly for primigravid (18.5%) and multigravid females (24.0%), though abortion tended to occur more frequently in primigravida. Perinatal mortality (16.1%) accounted for most of the losses under each housing condition. More successful pregnancies (90%) were recorded for females housed individually in type B cages than for females housed in type A cages (68%). About 50% of the couples originally established remained until weaning of their infants, yielding 77% viable offspring. For multigravid females statistical evaluation showed a significant effect of housing conditions on reproductive outcome (X2-test 0.01 less than P less than 0.05) that could be entirely attributed to low losses in females housed individually in type B cages. It is concluded that housing conditions can have a profound influence on reproductive success in cynomolgus monkeys.     

Field Evaluation of Amitus bennetti (Hymenoptera: Platygasteridae), a Parasitoid of Bemisia argentifolii (Hemiptera: Aleyrodidae), in Cotton and Bean

Field cage experiments were conducted in Riverside, California to quantify the impact of releases of the parasitoid Amitus bennetti Viggiani & Evans on mortality of the whitefly Bemisia argentifolii Bellows & Perring. Single-row 50-m-long plots were planted with either cotton or bean. Cages were erected over the plants in each row, and adult whiteflies were released into the cages. Approximately 10 days later, adult parasitoids were released. Marked individual whiteflies were scored every 4 days for 6 weeks. Paired life tables were then constructed from census data from release and control cages over a single whitefly generation. Total whitefly mortality in release cages (71% in bean, 61% in cotton) was significantly greater than in control cages (25% in bean, 34% in cotton). The marginal rate for mortality attributable directly to the parasitism was 0.535 in the bean plots and 0.201 in the cotton plots. In addition, other mortality was greater in the release plots, possibly reflecting death of parasitized hosts before larval parasitoids could complete development. Parasitism was the greatest mortality factor in the study.     

The Role of Wheel Running in the Coupling of Two Simultaneous Circadian Rhythms of Motor Activity in the Rat

Motor activity is among the non-photic stimuli that act on the internal clock. We have tested the role of motor activity in the circadian pattern of rats under conditions near the lower limits of entrainment, that induce circadian rhythm dissociation. Three groups of 8 rats each were used: a) rats kept individually in 25×25×15 cm cages, b) rats in 50×25×15 cm cages, and c) rats in 50×25×15 cm cages with access to a running wheel. All the rats were kept under light-dark cycles of 22 hours (T22, 11L:11D) for 50 days, after which they were transferred to constant darkness. All the rats without a running wheel showed a motor activity pattern with two statistically significant circadian rhythms in the periodogram of Sokolove and Bushell: one circadian component entrained by the LD cycle, and another free-running. The rats with access to a running wheel showed several patterns: 5 rats showed only one rhythm entrained to the LD cycle, 2 rats showed circadian rhythm dissociation, and 1 showed only a free running rhythm. We believe that the simultaneous manifestation of two circadian components reflects the functional dissociation of the oscillators population that constitutes the circadian pacemaker, of the rat. Physical exercise acts on the pacemaker reinforcing the strongest group of oscillators, which, depending on the structure of the circadian system of the rat, is usually the one entrained to the LD cycle. This study supports the hypothesis that motor activity couples the oscillators that form the circadian system of the rat.     

The Role of Wheel Running in the Coupling of Two Simultaneous Circadian Rhythms of Motor Activity in the Rat

Motor activity is among the non-photic stimuli that act on the internal clock. We have tested the role of motor activity in the circadian pattern of rats under conditions near the lower limits of entrainment, that induce circadian rhythm dissociation. Three groups of 8 rats each were used: a) rats kept individually in 25×25×15 cm cages, b) rats in 50×25×15 cm cages, and c) rats in 50×25×15 cm cages with access to a running wheel. All the rats were kept under light-dark cycles of 22 hours (T22, 11L:11D) for 50 days, after which they were transferred to constant darkness. All the rats without a running wheel showed a motor activity pattern with two statistically significant circadian rhythms in the periodogram of Sokolove and Bushell: one circadian component entrained by the LD cycle, and another free-running. The rats with access to a running wheel showed several patterns: 5 rats showed only one rhythm entrained to the LD cycle, 2 rats showed circadian rhythm dissociation, and 1 showed only a free running rhythm. We believe that the simultaneous manifestation of two circadian components reflects the functional dissociation of the oscillators population that constitutes the circadian pacemaker, of the rat. Physical exercise acts on the pacemaker reinforcing the strongest group of oscillators, which, depending on the structure of the circadian system of the rat, is usually the one entrained to the LD cycle. This study supports the hypothesis that motor activity couples the oscillators that form the circadian system of the rat.     

Laboratory and field evaluation of insect repellents as oviposition deterrents against the mosquito Aedes albopictus

Three experimental approaches were used to evaluate the oviposition deterrency of three insect repellents, AI3-35765, AI3-37220 (piperidine compounds), and the standard N,N-diethyl-3-methylbenzamide (deet) to the mosquito Aedes albopictus Skuse (Diptera: Culicidae). Against laboratory-reared Ae. albopictus gravid females, the EC50 values of AI3-37220, AI3-35765 and deet were 0.004%, 0.008% and 0.011% in laboratory cages and 0.004%, 0.01% and 0.009% in an outdoor screened cage. For a natural population of Ae. albopictus tested in the field, the EC50 values were determined as 0.004%, 0.008% and 0.001%, respectively. Ageing concentrations of 0.1% of each repellent provided >50% effective oviposition deterrency against the laboratory population of Ae. albopictus for 13 days in laboratory cages, for 15 days in the outdoor cage, and for 21 days against field population of Ae. albopictus in Florida. These topical skin repellents are effective oviposition deterrents for Ae. albopictus when employed at relatively low application rates.     

Predation and depth effects on abundance and size distribution of an invasive bivalve, the golden mussel Limnoperna fortunei, in a dam reservoir

We investigated the predation and depth effects on abundance and size distribution of an invasive freshwater bivalve, Limnoperna fortunei, post-larvae using screened (5-, 25-, and 50-mm mesh openings) and unscreened cages at three depths (6, 12, and 1 m above the bottom) in a dam reservoir, Lake Ohshio of Japan. The densities of L. fortunei on the unscreened cages were much lower than those of the 5-mm mesh screened cages at any depth. The predation rates estimated by dividing the density of the unscreened cage by that of the 5-mm mesh screened cage were 97.3% at 6 m depth, 96.6% at 12 m depth, and 95.8% at the bottom. Shell lengths of L. fortunei post-larvae were small on bottom cages, and their size distribution was significantly different from those at other depths. These results suggest that predation and depth affected the abundance and size distribution of L. fortunei in the lentic environment. The impact of L. fortunei invasion on the food web and the possibility of regulating their population by predation pressures are also discussed.     

The effect of salmon farming on the benthos of a Scottish sea loch

The effects of waste from a salmon farm on the benthos of a fjordic sea loch on the western coast of Scotland have been studied. Within 3 m of the floating cages the sediment was highly reducing, and dissolved oxygen content of the water overlying the sediment ranged from 35 to 75% saturation. At ⩾ 15 m from the cages the sediment was oxygenated, and the dissolved oxygen content of the water overlying the sediment was 50–85% saturation. Sedimentary redox potential and dissolved oxygen content of bottom water showed a seasonal variation. The benthic fauna showed marked changes in species number, species diversity, faunal abundance, and biomass in the region of the fish farm, with four zones of effect identified. Directly beneath, and up to the edge of the cages, there was an azoic zone. A highly enriched zone, dominated byCapitella capitata (Fabricius) andScolelepis fuliginosa (Claparède), occurred from the edge of the cages out to as 8 m. A slightly enriched ″transitional ″ zone occurred at ⩽ 25 m, and a “clean” zone at distances > 25 m. This study showed that salmon farming had similar effects on the benthos as other forms of organic enrichment, but the effects were limited to a small area in the immediate vicinity of the cages     

Phosphorylation of B-50 Protein by Calcium-Activated, Phospholipid-Dependent Protein Kinase and B-50 Protein Kinase

B-50 is a brain-specific phosphoprotein, the phosphorylation state of which may play a role in the regulation of (poly)phosphoinositide metabolism. Several kinases were tested for their ability to phosphorylate purified B-50 protein. Only calcium-activated, phospholipid-dependent protein kinase (kinase C) and B-50 protein kinase were able to use B-50 protein as a substrate. Furthermore, kinase C specifically phosphorylates B-50 when added to synaptic plasma membranes. We further characterized the sensitivity of kinase C and B-50 kinase to ACTH (and various fragments), phospholipids, chlorpromazine, and proteolytic activation. Since the sensitivities of both kinases were similar, we conclude that B-50 protein kinase is a calcium-dependent, phospholipid-stimulated protein kinase of the same type as kinase C.     

单、双枚椎间融合器在单节段腰椎后路融合术中的应用效果比较

目的:比较腰椎后路融合术(PLIF)单、双枚椎间融合器(Cage)的使用对远期疗效的影响。方法:回顾我院2004-2011年间164例因腰椎退变疾病行单节段PLIF患者的临床资料,按Cage数目将其分为单Cage组(114例)与双Cage组(50例)。获得其住院资料和术后2年以上临床与影像学随访资料并比较分析。结果:平均随访时间47.5个月。两组间的年龄、性别、术前JOA评分、术后随访时间的差别无显著性(P0.05),但单Cage组失血量更低(P0.05),但随访时相对椎间隙高度低于双Cage组(P0.05);而两组间JOA评分改善率、随访时JOA评分、优良率、手术耗时、住院天数、并发症发生率的差别无显著性(P0.05)。结论:PLIF中单、双Cage的使用均能获得满意的疗效与安全性,与双Cage相比,单Cage手术失血更少,但术后远期相对椎间隙高度更低,对于这类患者应加强随访并警惕断钉等并发症的发生。     

Dispersal of Steinernema glaseri (Nematoda: Steinernematidae) in adult Japanese beetles, Popillia japonica (Coleoptera: Scarabaeidae)

Japanese beetle adults, Popillia japonica, can become infected with and disperse the entomopathogenic nematode, Steinernema glaseri, under laboratory and field conditions. After a 24-h exposure to 10 000 infective juveniles/20 adult beetles, 45% of the beetles died within 4 days post-treatment, but only 59% of these were infected with the nematode. Corresponding control mortality was 6.5%. An average of 238 infective juveniles were produced/beetle. Beetles exposed to 4000 and 10 000 infectives/10 adults carried with them an average of 17 and 59 infectives/adult on external body surfaces respectively. When beetles that had been exposed to 4000 infectives/20 adults were transferred to, and held in, cages containing soil for 2 weeks, up to 89% of the adults died, as did 74% of the P. japonica larvae that were subsequently placed in the cages. When adults that had been exposed to 50 000 infectives/250 beetles in moist sand for 16 h were released into screened cages in the field at soil temperatures of over 25 C, the soil beneath 83% of the cages tested positive for the nematode, using Galleria mellonella larvae as bait, 2 weeks after releasing the beetles. No nematodes were detected in control plots. The potential of infected adult P. japonica for dispersing S. glaseri by flight was investigated by exposing adults to 50 000 infectives/250 beetles, marking and releasing them in the field and recapturing them in lure-baited Japanese beetle traps. Less than 1% of the treated beetles were recaptured, but 33% of these had one or more nematodes in their hemocoels. Accordingly, this approach does not appear to be feasible for large-scale augmentation and dispersal of the nematode using currently developed methods of infection. If improvements in mass-inoculation methods can be made that enable a rapid high percentage of infection while still permitting flight, this concept could be employed to establish new foci of infection or for the introduction of other species of nematodes.     

Mosquito density, biting rate and cage size effects on repellent tests

Mosquito biting rates and the mean duration of protection (in hours) from bites (MDPB) of Aedes aegypti and Anopheles quadrimaculatus , using the repellent 'deet' ( N , N -diethyl-3-methylbenzamide) on a 50 cm² area of healthy human skin, were observed in small (27 l), medium (≈65 l) and large (125 l) cages containing low, medium or high densities of mosquitoes: respectively, 640, 128 or 49 cm³ of cage volume per female. At the initial treatment rate of ≈ 0.4 μl/cm² (1 ml of 25% deet in ethanol on 650 cm² of skin), the MDPB for deet against Ae. aegypti ranged from 4.5 to 6.5 h and was significantly less (5.0 ± 0.8 h) in large cages compared with medium (6.2 ± 0.9 h) and small (6.2 ± 0.8 h) cages, regardless of the density. Against An. quadrimaculatus the MDPB for deet 0.4 μl/cm² was 1.5–8.0 h, less in small (3.7 ± 2.3 h) and large (2.2 ± 1.1 h) cages at medium (3.7 ± 2.3 h) and high (2.5 ± 1.7 h) mosquito densities, and was longest in medium cages (6.2 ± 2.6 h) at low mosquito densities (5.8 ± 2.8 h). With equinoxial photoperiodicity (light on 06.00–18.00 hours) the biting rate was influenced by the time of observation (08.00, 12.00, 16.00 hours) for Ae. aegypti but not for An. quadrimaculatus. For both species, the biting rate was inversely proportional to mosquito density and the MDPB. The shortest MDPBs were obtained in large cages with high densities of mosquitoes and longest protection times occurred in medium sized cages with low mosquito densities.