The contractile and regulatory proteins of insect flight muscle

1. Myosin, actin and the regulatory proteins were prepared from insect flight muscle. 2. The light subunit composition of the myosin differed from that of vertebrate muscle myosin. The ionic strength and pH dependence of the myosin adenosine triphosphatase (ATPase) were measured. 3. Actin was associated with a protein of subunit molecular weight 55000 and was purified by gel filtration. Impure actin had protein bound at a periodicity of about 40nm. 4. Regulatory protein extracts had tropomyosin and troponin components of subunit molecular weight 18000, 27000 and 30000. Crude extracts of regulatory proteins inhibited the ATPase activity of desensitized or synthetic actomyosin; this inhibition was relatively insensitive to high Ca(2+) concentrations. Purified insect regulatory protein produced as much sensitivity to Ca(2+) as did the rabbit troponin-tropomyosin complex. 5. Synthetic actomyosins were made from rabbit and insect proteins. Actomyosins containing insect myosin had a low ATPase activity that was activated by tropomyosin. The Ca(2+) sensitivity of actomyosins containing insect myosin or actin, with added troponin-tropomyosin complex from rabbit, was comparable with that of rabbit actomyosin.     

First case of rabbit haemorrhagic disease in Canada: contaminated flying insect, vs. long-term infection hypothesis

Following the announcement of the first case of rabbit haemorrhagic disease (RHD) in a pet rabbit, housed indoors in Canada for more than 1 year, I submitted an evidence‐based explanation to ProMed explaining how RHD might have caused the death of ‘one’ of the three pet rabbits. I suggested with supporting evidence, that it may have been persistently infected with rabbit haemorrhagic disease virus (RHDV) which may have reactivated to cause the fatal disease. However, in this issue, Peacock et al. have proposed an alternative ‘hypothesis’ for the appearance of RHD in the pet rabbit. They hypothesise that a non‐identified insect or fomite might have become contaminated by a Chinese strain of RHDV somewhere in the US. This insect/fomite then flew or was windborne, from the US to Canada where it entered the house containing three pet rabbits and infected one of them. RHD is non‐endemic and is rarely reported in the US, where it has only been observed in domestic European rabbits, held in rabbitries. My proposal was based on the details provided by ProMed, the veterinary report from Canada, where RHDV has never previously been identified and the epidemiological, ecological and evolutionary history of RHDV which includes serological and phylogenetic evidence that ancestral RHDV lineages circulated before 1984. The flying insect hypothesis of Peacock et al. is based on circumstantial evidence and, I believe, has a lower probability of being correct than my evidence‐based long‐term infection proposal.     

Structural and functional properties of Drosophila melanogaster phosphorylase: comparison with the rabbit skeletal muscle enzyme

Glycogen phosphorylase isolated from Drosophila melanogaster contains one pyridoxal 5'-phosphate per subunit; the coenzyme is in a hydrophobic environment. Fruit-fly phosphorylase a has lower KM for glucose-1-phosphate and is less sensitive to allosteric inhibitors than the b form of the enzyme. The amino acid composition of Drosophila phosphorylase differs from that of rabbit skeletal muscle phosphorylase. These two enzymes give distinct one dimensional peptide maps. The distribution of reactive SH-groups is markedly different in the insect and vertebrate phosphorylase. Fruit-fly phosphorylase a is dephosphorylated by either rabbit or Drosophila protein phosphatase-1 at a slower rate than rabbit muscle phosphorylase a.     

Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection.

VP60, the unique component of rabbit hemorrhagic disease virus capsid, was expressed in the baculovirus system. The recombinant VP60, released in the supernatant of infected insect cells, assembled without the need of any other viral component to form viruslike particles (VLPs), structurally and immunologically indistinguishable from the rabbit hemorrhagic disease virion. Intramuscular vaccination of rabbits with the VLPs conferred complete protection in 15 days; this protection was found to be effective from the fifth day after VLP injection and was accompanied by a strong humoral response.     

Calcium-Dependent Myosin from Insect Flight Muscles

Calcium regulation of the insect actomyosin ATPase is associated with the thin filaments as in vertebrate muscles, and also with the myosin molecule as in mollusks. This dual regulation is demonstrated using combinations of locust thin filaments with rabbit myosin and locust myosin with rabbit actin; in each case the ATPase of the hybrid actomyosin is calcium dependent. The two regulatory systems are synergistic, the calcium dependency of the locust actomyosin ATPase being at least 10 times that of the hybrid actomyosins described above. Likewise Lethocerus myosin also contains regulatory proteins. The ATPase activity of Lethocerus myosin is labile and is stabilized by the presence of rabbit actin. Tropomyosin activates the ATPase of insect actomyosin and the activation occurs irrespective of whether the myosin is calcium dependent or rendered independent of calcium.     

Two independent pathways of expression lead to self-assembly of the rabbit hemorrhagic disease virus capsid protein.

The rabbit hemorrhagic disease virus capsid protein was expressed in insect cells either as an individual protein species, from a mRNA analogous to the viral subgenomic RNA, or as part of a polyprotein that included the viral 3C-like protease and the RNA polymerase. Both pathways of expression led to the assembly of viruslike particles morphologically and antigenically similar to purified virus.     

Identification of Epitope on DNA-binding Protein Expressed in Insect Cell Infected by Baculovirus

DNA-binding protein (DBP) is an early gene product produced during viral replication. Polyclonal anti-DBP was produced using rabbit by intradermal injections of Escherichia coli-expressed purified recombinant DBP. Prepared anti-DBP completely blocked the replication of baculovirus in insect cells. The anti-DBP binding to DBP was confirmed by both Western blotting with Tn-5B1-4 insect cell lysates as well as immunostained baculovirus-infected Tn-5B1-4 insect cells. To determine the anti-DBP epitope 12 peptides were synthesized and their specific-binding activities were measured using ELISA. Based on specific-binding activity against anti-DBP the epitope was predicted to be between amino acid residues 248–265 (QRMSVEDFDRLFEMDKID). Especially from 18 amino acid residues it was further to be narrowed between amino acid residues 260–265 (EMDKID) which showed a critical role in specific-binding activity.     

Seasonality of insect succession on exposed rabbit carrion in Guangzhou, China

Abstract  Succession patterns for insect fauna, spanning four seasons, on rabbit carcasses were studied in Guangzhou China from 2006 to 2008. A total of 49 taxa belonging to three orders and 15 families were collected and identified. Four decomposition stages were recognized: fresh, bloated, decay and dry stage. Taxa diversity was higher in bloated and decay stages than fresh and dry stages. Internal and ambient temperatures were measured. Maximum internal temperature arrived earlier in warmer seasons. The sequence of insect succession in this study follows the same general patterns: the Diptera peaked initially, and the Coleoptera peaked later. Chrysomya megacephala was the dominant species in four seasons. Its time point of different development stages on carrion could be the most important indicators for PMI (postmortem interval) estimation in Guangzhou. Yearly and seasonal differences in activities of insects were compared. Alpha diversity indices were calculated. There were no significant differences in Shannon-Wiener index, evenness ( J ) and dominance ( C ) between seasons and years. From the Jaccard similarity index, the highest similarity was between winter and spring, while the lowest was between summer and winter. Some species occurring exclusively in one season may be important to characterize different seasons. A list of the insect taxa associated with above-ground rabbit carrion during the four seasons was compiled, which can be used for estimating time since death in similar bio-climate areas.     

Contrasting effects of insect and molluscan herbivores on plant diversity in a long-term field experiment

The importance of invertebrate herbivores in regulating plant communities remains unclear, due to the absence of long-term exclusion experiments. An experiment in an English grassland involving long-term exclusions of insect and mollusc herbivores, along with rabbit fencing, showed strong, but opposing, effects of the invertebrate herbivores. Plant species richness declined and biomass increased following insect exclusion, due to increased dominance by a grass species, whereas mollusc exclusion led to increased herbs abundance. The two herbivores had a compensatory interaction: molluscs had no effects in the absence of insects and large insect effects depended on the absence of molluscs. The effects of invertebrate exclusion became apparent only after 8 years, and would have been seriously underestimated in shorter studies. Our results suggest that theorists and conservation managers need to shift from their historic focus on vertebrate herbivory, to a recognition that invertebrates can be equally important drivers of plant community structure.     

The crystal structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster at 2.5 A resolution

The structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster has been determined by X-ray diffraction at 2.5 A resolution. The insect enzyme crystallizes in space group P2(1)2(1)2(1) with lattice replacement with rabbit muscle aldolase as a search model has been employed to solve the structure. To improve the initial phases real space averaging, including phase extension from 4.0 to 2.5 A, has been applied. Refinement of the atomic positions by molecular dynamics resulted in a crystallographic R-factor of 0.214. The tertiary structure resembles in most parts that of the vertebrate aldolase from rabbit muscle. Significant differences were found in surface loops and the N- and C-terminal regions of the protein. Here we present the first aldolase structure where the functionally important C-terminal arm is described completely.     

Impacts of antemortem ingestion of alcoholic beverages on insect successional patterns

This paper studies the impacts of antemortem ingestion of alcoholic beverages by the domestic rabbit, Oryctolagus cuniculus L., on postmortem successional patterns of insects during winter and summer 2018 in Riyadh, Saudi Arabia. Insect samples were collected from the carcasses of rabbits fed alcoholic beverages as well as untreated rabbits for 15 days postmortem during two successional studies in each season. The results showed that, during both seasons, the decomposition process for the carcasses of rabbits fed alcoholic beverages antemortem was one to two days longer. The results also showed, however, that alcoholic beverages did not affect insect succession patterns in either season. In fact, the number of insects appeared to be influenced by the ambient temperature during the two seasons, with 4415 insects in the winter compared to 1033 insects in the summer. In total, 30 insect taxa were collected during the winter study from the carcasses of rabbits fed alcoholic beverages antemortem; while 26 of these same taxa were collected from the carcasses of the untreated rabbits. Among the treated rabbits, those fed 25 ml alcoholic beverages treated attracted the highest number of insect taxa (24 taxa). In the summer study, 21 insect taxa were collected in total, 19 from the carcasses of the alcohol-treated rabbits and 13 from untreated rabbits. Among the treated rabbits, those fed 50 ml alcoholic beverages attracted the highest number of insect taxa (14 taxa). These results contribute to the understanding of the factors affecting the use of insects in medical investigations, given that alcoholic beverages are a common addictive agent.     

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.     

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institues of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749. from the National Institutes of Health.     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata, andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749 from the National Institutes of Health.     

Hybridoma antibodies as specific probes to Drosophila melanogaster yolk polypeptides

Hybridoma antibodies to Drosophila melanogaster soluble yolk proteins (YPs) were developed by both in vivo and in vitro immunizations followed by the fusion of SP2/0-Ag14 cells and splenocytes of BALB/c mice. Rabbit antiserum was made female specific by affinity column with male proteins as ligand. The binding sites of these hybridoma antibodies and rabbit antibodies towards different YP components were identified with a combination of gel electrophoresis, Western blotting and immunohistochemical staining. A double antibody sandwich enzyme-linked immunosorbent assay was developed with monoclonal antibodies from 2 cell lines and alkaline phosphatase labelled rabbit polyclonal antibodies as primary and secondary antibodies respectively. Yolk polypeptide levels in the haemolymph can be monitored in individual insect samples.     

昆虫细胞中存在角蛋白纤维

本文采用细胞分级抽提结合整装细胞电镜制样技术,分别在两种昆虫细胞:斜纹夜蛾(SL)细胞;甜菜夜蛾(SE)细胞中显示了一个精细的中等纤维网络结构,纤维自胞核发出,排列错综复杂,其单丝清晰可见,直径约为8～10nm;间接免疫荧光染色结果表明角蛋白抗体在两种细胞中均能显示出清晰的荧光纤维网络,而且荧光纤维的分布有所不同;用角蛋白抗体对这两种细胞全蛋白进行免疫印迹实验,均可显示49KD,68KD的两个主要多肽条带,说明这两种昆虫细胞中等纤维的主要成分为角蛋白.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for "native" lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Regulation of muscle phosphorylase b kinase activity by inorganic phosphate and calcium ions

The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P(i) and Ca(2+) was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P(i) increased activity severalfold. The effect was concentration-dependent, with an apparent K(m) of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg(2+) in addition to P(i). The apparent K(m) for Mg(2+) was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P(i), and was strongly inhibited by K(+), Na(+) and Li(+). At physiological concentrations, Ca(2+) activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca(2+) of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca(2+), and showed a large increase in activity over a narrow range of Ca(2+) concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca(2+), and had a smaller increase over a wide range of Ca(2+) concentration. The concentrations of Ca(2+) required for half-activation were 0.1 and 1mum for the blowfly and rabbit enzymes respectively. The pH-activity profiles of the non-activated, phosphate- and Ca(2+)-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.     

Arthrin: a new actin-like protein in insect flight muscle

There are one or more proteins of 50,000 to 60,000 Mr in the thin filaments of insect flight muscle. A protein of 55,000 Mr has been isolated from insect fibrillar flight muscle and called arthrin. Despite its higher molecular weight, arthrin is in many ways like actin. The amino acid composition of arthrin was similar to that of actin. There were similarities in the peptides produced by digesting the denatured proteins and mild digestion of polymerized proteins cleaved similar-sized fragments from arthrin and actin. Polymerized arthrin activated the Mg2+ ATPase of myosin to the same extent as actin and the ATPase was regulated by rabbit or Lethocerus troponin and tropomyosin. Arthrin did not itself act as troponin-T. Electron microscopy of negatively stained specimens showed that arthrin and actin filaments were similar in structure and that arthrin could be decorated by rabbit subfragment-1 to form normal-looking arrowheads. Arthrin formed paracrystals at an optimum concentration of MgCl2 (25 mM) that was somewhat lower than the optimum for actin paracrystals. Optical diffraction showed that the structure of the paracrystals was similar to those formed from actin. The mass of arthrin and actin filaments relative to phage fd was measured by scanning transmission electron microscopy; the relative mass of arthrin and actin was 1.33, in agreement with molecular weight estimations. Therefore arthrin has the properties of a heavy form of actin. The proportion of actin, arthrin and troponin-T in Lethocerus myofibrils was six moles of actin to one mole of arthrin and one mole of troponin-T. The function of arthrin is not known.