Functional conservation of clock output signaling between flies and intertidal crabs

Intertidal species have both circadian and circatidal clocks. Although the behavioral evidence for these oscillators is more than 5 decades old, virtually nothing is known about their molecular clockwork. Pigment-dispersing hormones (PDHs) were originally described in crustaceans. Their insect homologs, pigment-dispersing factors (PDFs), have a prominent role as clock output and synchronizing signals released from clock neurons. We show that gene duplication in crabs has led to two PDH genes (β-pdh-I and β-pdh-II). Phylogenetically, β-pdh-I is more closely related to insect pdf than to β-pdh-II, and we hypothesized that β-PDH-I may represent a canonical clock output signal. Accordingly, β-PDH-I expression in the brain of the intertidal crab Cancer productus is similar to that of PDF in Drosophila melanogaster, and neurons that express PDH-I also show CYCLE-like immunoreactivity. Using D. melanogaster pdf-null mutants (pdf(01)) as a heterologous system, we show that β-pdh-I is indistinguishable from pdf in its ability to rescue the mutant arrhythmic phenotype, but β-pdh-II fails to restore the wild-type phenotype. Application of the three peptides to explanted brains shows that PDF and β-PDH-I are equally effective in inducing the signal transduction cascade of the PDF receptor, but β-PDH-II fails to induce a normal cascade. Our results represent the first functional characterization of a putative molecular clock output in an intertidal species and may provide a critical step towards the characterization of molecular components of biological clocks in intertidal organisms.     

Cloning of the DNA fragment of a transgenic mouse containing an integrated recombinant plasmid

The "plasmid rescue" method has been used to isolate the recombinant plasmid pMA3 fragment and flanking sequences from the transgenic mouse genome containing the fragment in integrated form. The "rescued" plasmid, pMAR1, lacks all virus sequences and retains only those regions of pBR322 that are responsible for the plasmid replication and Escherichia coli ampicillin resistance. The plasmid pMA3 deletion has occurred at its integration into the mouse genome after microinjection into the zygote. The integrated fragment of the plasmid is adjacent to the genome repeated sequence that is highly conservative in evolution.     

On the coordination of motor output during visual flight control of flies

Summary In tethered flying houseflies (Musca domestica), the yaw torque produced by the wings is accompanied by postural changes of the abdomen and hindlegs. In free flight, these body movements would jointly lead to turning manoeuvres of the animal. By recording the yaw torque together with the lateral deflections of either the abdomen or the hindlegs, it is shown that these motor output systems act in a highly synergistic way during two types of visual orientation behavior, compensatory optomotor turning reactions and orientation turns elicited by moving objects. This high degree of coordination is particularly conspicuous for the pathway activated by moving objects. Here, orientation responses either may be induced or may fail to be generated always simultaneously in all three motor output systems. This suggests that the pathway mediating orientation turns towards objects is gated before it segregates into the respective motor control systems of the wings, the abdomen and the hindlegs.     

HBV转基因小鼠--乙型肝炎研究的重要工具

HBV感染具有严格的种属特异性和组织亲嗜性,所以可用于研究的动物模型很少。随着转基因技术的出现和发展,人们通过研制转基因动物模型来研究病毒致病机理。把HBV的DNA或其片段转入小鼠受精卵建立起来的HBV转基因小鼠,已被广泛地应用于乙型肝炎的研究中,主要体现在以下3个方面：研究HBV的致病机制、与肝细胞癌的关系及寻找、评价治疗乙肝的方法。     

cAMP-dependent protein kinase and the disruption of learning in transgenic flies.

A molecular genetic approach was used to test for a role of cAMP-dependent protein kinase (PKA) in learning and memory in Drosophila. We used genes encoding a peptide inhibitor of PKA, an N-terminal regulatory subunit fragment containing a pseudosubstrate inhibitory domain, and a wild-type catalytic subunit. These dominantly acting genes were placed under control of the hsp70 promoter and transformed into wild-type flies. Induction of the transgenes by 1 hr heat shock results in overproduction of their RNA in adult flies. The same heat shock treatment disrupts the ability of the flies to learn in an odor discrimination task reinforced with electric shock. The results demonstrate the involvement of PKA in the associative learning of Drosophila.     

Case study: data management strategies in an integrated pathway tool

This paper describes the development strategies for an integrated tool to support scientists in the creative exploration of data relating to biochemical pathways. The multiple user groups, diverse functionalities, and many types and sources of data demanded a flexible yet coherent approach. This paper summarises the software requirements and the implied modules and functions, and focuses on the design decisions relevant to the representation, management and flow of data. Finally, several case studies in the use of the software are described and evaluated, and recommendations are made for future work.     

Properties of a motor output system involved in the optomotor response in flies

1. The optomotor response of tethered flying houseflies (Musca domestica) has been studied at the level of the neural output which controls the activities of some non-fibrillar flight muscles (N-muscles).-a) During visually induced turning responses in a given direction some N-muscles on the right side of the thorax are synergistically active together with other N-muscles on the left side of the thorax. The same muscles are inactive during turning reactions in the opposite direction while the corresponding antagonists are now active (synopsis in Table 1).-b) The response activities of the N-mussles show a considerable variation during the course of time in spite of constant visual input.-c) There is a strong tendency for N-muscle spikes to be phase-locked with respect to the wingbeat period.-d) The findings obtained fromMusca are in accordance with the corresponding results obtained fromCalliphora (Heide, 1971b).
2. TheN-muscle activities have also been investigated in tethered flying blowflies (Calliphora erythrocephala) which tried to yaw spontaneously with both wings beating. In spontaneous left (right) turn reactions the features of the observed neural output are nearly identical with the features of the motor output showing up during visually induced left (right) turn reactions.-A different motor output pattern has been found in flies with only one wing beating.
3. The wingbeat synchronous rhythm observed in spike trains from activeN-muscles is produced in the thorax without the participation of higher stages of the fly's CNS. On the other hand no distinct rhythms can be found in spike trains fromN-muscles of non-flying flies when their motoneurons are artificially activated by non-rhythmic stimuli. Afferent information from thoracic sense organs seems to be essential for the production of the rhythm observed during flight.
4. The results about the production of the wingbeat synchronous rhythm in spike trains fromN-muscles suggest that the information derived from the motion detectors only acts to gate the output needed to achieve yaw-turn reactions. The strength of the influence of signals from the motion detectors on the output producing system can be modified by the animals “state of excitement”.
5. A model is presented which summarizes some features of information processing in the output systems supplying theN-muscles of flies. Available physiological data are discussed in relation to the model.

     

Overexpression of biglycan in the heart of transgenic mice: an antibody microarray study

Biglycan, a small leucine rich proteoglycan, is expressed in almost every tissue of the body, mainly in the extracellular matrix of connective tissues. Although there is an increasing amount of data on the biological role of biglycan protein, its function is still poorly understood. We aimed to gather more information about the biological function of biglycan protein in the cardiac tissues, and its role in signal transduction. Therefore, we generated transgenic mice overexpressing the human biglycan protein and analyzed the cardiac protein profile of transgenic offsprings using quantitative real-time (QRT)-PCR and proteomics. QRT-PCR results showed that most members of extracellular matrix were downregulated whereas cadherins, TGF-beta1, and TGF-beta2 were upregulated. Antibody microarrayer experiment revealed that pyk2, RAF-1, Mcl-1, syntrophin, calmodulin, isoforms of NOS protein family (eNOS, nNOS, and iNOS), and synaptotagmin proteins were unambiguously upregulated in the heart of biglycan transgenic mice. In this study we show that biglycan directly or indirectly activates proteins involved in cardiac remodeling (TGF-beta, pyk2), signal transduction (RAF-1, Mcl-1, syntrophin, calmodulin, nNOS p38MAPK and MAP kinases), cardioprotection (NOS family, TGF-beta) and Ca++ signaling (connexin, calmodulin, synaptotagmin). On the basis of the results presented here, we conclude that biglycan is a multifunctional extracellular protein that has a pivotal role in pathological remodeling of cardiac tissue and mediates cardioprotection.     

Stretch-shortening cycle exercises: an effective training paradigm to enhance power output of human single muscle fibers.

Functional performance of lower limb muscles and contractile properties of chemically skinned single muscle fibers were evaluated before and after 8 wk of maximal effort stretch-shortening cycle (SSC) exercise training. Muscle biopsies were obtained from the vastus lateralis of eight men before and after the training period. Fibers were evaluated regarding their mechanical properties and subsequently classified according to their myosin heavy chain content (SDS-PAGE). After training, maximal leg extensor muscle force and vertical jump performance were improved 12% (P<0.01) and 13% (P<0.001), respectively. Single-fiber cross-sectional area increased 23% in type I (P<0.01), 22% in type IIa (P<0.001), and 30% in type IIa/IIx fibers (P<0.001). Peak force increased 19% in type I (P<0.01), 15% in type IIa (P<0.001), and 16% in type IIa/IIx fibers (P<0.001). When peak force was normalized with cross-sectional area, no changes were found for any fiber type. Maximal shortening velocity was increased 18, 29, and 22% in type I, IIa, and hybrid IIa/IIx fibers, respectively (P<0.001). Peak power was enhanced in all fiber types, and normalized peak power improved 9% in type IIa fibers (P<0.05). Fiber tension on passive stretch increased in IIa/IIx fibers only (P<0.05). In conclusion, short-term SSC exercise training enhanced single-fiber contraction performance via force and contraction velocity in type I, IIa, and IIa/IIx fibers. These results suggest that SSC exercises are an effective training approach to improve fiber force, contraction velocity, and therefore power.     

Effect of interrepetition rest on power output in the power clean

The effect of interrepetition rest (IRR) periods on power output during performance of multiple sets of power cleans is unknown. It is possible that IRR periods may attenuate the decrease in power output commonly observed within multiple sets. This may be of benefit for maximizing improvements in power with training. This investigation involved 10 college-aged men with proficiency in weightlifting. The subjects performed 3 sets of 6 repetitions of power cleans at 80% of their 1 repetition maximum with 0 (P0), 20 (P20), or 40 seconds (P40) of IRR. Each protocol (P0, P20, P40) was performed in a randomized order on different days each separated by at least 72 hours. The subjects performed the power cleans while standing on a force plate with 2 linear position transducers attached to the bar. Peak power, force, and velocity were obtained for each repetition and set. Peak power significantly decreased by 15.7% during P0 in comparison with a decrease of 5.5% (R1: 4,303 ± 567 W, R6: 4,055 ± 582 W) during P20 and a decrease of 3.3% (R1: 4,549 ± 659 W, R6: 4,363 ± 476 W) during P40. Peak force significantly decreased by 7.3% (R1: 2,861 ± 247 N, R6: 2,657 ± 225 N) during P0 in comparison with a decrease of 2.7% (R1: 2,811 ± 327 N, R6: 2,730 ± 285 N) during P20 and an increase of 0.4% (R1: 2,861 ± 323 N, R6: 2,862 ± 280 N) during P40. Peak velocity significantly decreased by 10.2% (R1: 1.97 ± 0.15 m·s(-1), R6: 1.79 ± 0.11 m·s(-1)) during P0 in comparison with a decrease of 3.8% (R1: 1.89 ± 0.13 m·s(-1), R6: 1.82 ± 0.12 m·s(-1)) during P20 and a decrease of 1.7% (R1: 1.93 ± 0.17 m·s(-1), R6: 1.89 ± 0.14 m·s(-1)) during P40. The results demonstrate that IRR periods allow for the maintenance of power in the power clean during a multiple set exercise protocol and that this may have implications for improved training adaptations.     

Creation of low-copy integrated transgenic lines in Caenorhabditis elegans

In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing.