Protein kinase C signalling involved in prothoracicotropic hormone-stimulated prothoracic glands in the silkworm, Bombyx mori.

In the present study, the participation of protein kinase C (PKC) signalling in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx prothoracic glands (PGs) is demonstrated and characterized. PTTH stimulated phosphorylation of a 37-kDa protein in Bombyx PGs both in vitro and in vivo, as recognized by a PKC substrate antibody. Treatment with either A23187 or thapsigargin also stimulated this 37-kDa protein phosphorylation. PTTH-stimulated phosphorylation of the 37-kDa protein was markedly attenuated in the absence of Ca2+ . The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH-stimulated phosphorylation of this protein, indicating the involvement of Ca2+ and PLC. A mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (U0126), a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) and a chemical activator of adenosine 5'-monophosphate-activated protein kinase (AMPK) (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside) did not affect PTTH-stimulated phosphorylation of the 37-kDa protein, implying that ERK and PI3K/AMPK are not the upstream signalling pathways for PKC-dependent protein phosphorylation. The mitochondrial oxidative phosphorylation inhibitors (the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone and diphenylene iodonium) inhibited PTTH-stimulated phosphorylation of the 37-kDa protein, indicating its redox regulation. Treatment with PKC inhibitors (either calphostin C, chelerythrine C or rottlerin) reduced PTTH-stimulated phosphorylation of the 37-kDa protein. PTTH-stimulated ecdysteroidogenesis was also inhibited by treatment with rottlerin, thus further confirming participation of PKC-dependent phosphorylation in PTTH signalling. From these results, we demonstrated that redox-regulated PTTH-stimulated PKC signalling is involved in ecdysteroid secretion in Bombyx PGs.

Involvement of RSK phosphorylation in PTTH-stimulated ecdysone secretion in prothoracic glands of the silkworm, Bombyx mori.

It is well known that phosphorylation of extracellular signal-regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signalling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage- and dose-dependent. The absence of Ca2+ reduced PTTH-stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH-stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH-regulated RSK phosphorylation, indicating its redox regulation. A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, decreased PTTH-stimulated RSK phosphorylation, indicating that ERK is an upstream signalling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH-stimulated RSK phosphorylation, and a PKC activator, phorbol 12-myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI-D1870, a specific RSK inhibitor, partly prevented PTTH-stimulated RSK phosphorylation and significantly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH-stimulated ecdysteroidogenesis.

Characterization and structure of glyceraldehyde-3-phosphate dehydrogenase type 1 from Escherichia coli.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that catalyzes the conversion of D-glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. Here, the full-length GAPDH type 1 from Escherichia coli (EcGAPDH1) was cloned and overexpressed, and the protein was purified. Biochemical analyses found that the optimum reaction temperature and pH of EcGAPDH1 were 55°C and 10.0, respectively. The protein has a certain amount of thermostability. Crystals of EcGAPDH1 were obtained using the sitting-drop vapor-diffusion technique and X-ray diffraction data were collected to 1.88 Šresolution. Characterization of the crystals showed that they belonged to space group P41212, with unit-cell parameters a = b = 89.651, c = 341.007 Å, α = ß = γ = 90°. The structure of EcGAPDH1 contains four subunits, each of which includes an N-terminal NAD+-binding domain and a C-terminal catalytic domain. Analysis of the NAD+-bound form showed some differences between the structures of EcGAPDH1 and human GAPDH. As EcGAPDH1 shares 100% identity with GAPDH from Shigella sonnei, its structure may help in finding a drug for the treatment of shigellosis.

[Investigation of a cluster epidemic of COVID-19 in Ningbo].

Objective: To investigate a cluster epidemic of COVID-19 after a mass gathering activity in Ningbo of Zhejiang province and analyze the transmission chain and status of infection cases of different generations. Methods: The tracking of all the close contacts of the first COVID-19 case and epidemiological investigation were conducted on January 29, 2020 after a cluster epidemic of COVID-19 related with a Buddhism rally on January 19 (the 1.19 rally) in Ningbo occurred. The swabs of nose/throat of the cases and close contacts were collected and tested for nucleic acids by real-time fluorescence quantitative RT-PCR. Results: From January 26 to February 20, 2020, a total of 67 COVID-19 cases and 15 asymptomatic infection cases related with the 1.19 rally were reported in Ningbo. The initial case was the infection source who infected 29 second generation cases and 4 asymptomatic infection cases, in whom 23 second generation cases and 3 asymptomatic infection cases once took bus with the initial case, the attack rate was 33.82% (23/68) and the infection rate was 38.24% (26/68). The risks of suffering from COVID-19 and being infected were 28.91 times and 26.01 times higher in rally participants taking bus with initial case compared with those taking no bus with initial case. In this epidemic, 37 third+generation cases and 11 related asymptomatic infection cases occurred, the attack rate was 2.88% (37/1 283) and the infection rate was 4.76% (48/1 008). The main transmission routes included vehicle sharing and family transmission. Conclusion: It was a cluster epidemic of COVID-19 caused by a super spreader in a massive rally. The epidemic has been under effective control.

[Study on transmission dynamic of 15 clusters of COVID-2019 cases in Ningbo].

Objective: To describe the basic characteristics of clusters of coronavirus diseases 2019 (COVID-19) cases in Ningbo, Zhejiang province, and evaluate the generation time (Tg) and basic reproduction number (R(0)) of COVID-19. Methods: The basic information and onset times of the clusters of COVID-19 cases in Ningbo were investigated, the inter-generational interval of the cases were fitted by using gamma distribution, and the R(0) was calculated based on the SEIR model. Results: In the 15 clusters of COVID-19 cases, a total of 52 confirmed cases, 5 cases of nucleic acid-positive asymptomatic cases. The cases occurred from January 23 to February 4, the cases were mainly women. The incubation period was (6.11±3.38) days, and the median was 5 days. The Tg was (6.93±3.70) days. There were no significant differences in Tg between age group<60 years and age group 60 years and above, and between men and women (P=0.551). According to the Tg calculated in this paper, the R(0) of COVID-19 in Ningbo was 3.06 (95%CI: 2.64- 3.51); according to the reported case transmission interval of 7.5 days in the literature, the R(0) was 3.32 (95%CI: 2.51-9.38). Conclusion: There is no age and gender specific differences in the Tg of clusters of COVID-19 cases in Ningbo, and COVID-19 has high infectivity and spreading power in early phase.

[The impact of ambient PM(2.5) on daily outpatient visits due to chronic obstructive pulmonary disease, among the urban residents of Ningbo city].

Objective: To explore the short-term effects of ambient PM(2.5) on the outpatient visits of chronic obstructive pulmonary disease (COPD) in Ningbo city. Methods: Through the regional health information platform, number of daily COPD outpatients from the four general hospitals in Ningbo was gathered. Related data on meteorological and air pollution from 2014 to 2016 was also collected. Generalized additive model (GAM) of Possion regression was used to estimate the impact of PM(2.5) pollution on COPD outpatients and the lagging effects. Results: In cold (November- April) or warm seasons (May-October), an 10 µg/m(3) increase of PM(2.5) would result in the excessive number of COPD outpatients as 1.87% (95%CI: 0.98%-2.76%), 2.09% (95%CI: 1.11%-3.08%) and 2.56% (95%CI: 0.56%-4.59%), respectively. In terms of the short-term effects of PM(2.5) the strongest was seen in the days of warm season but without delay (P<0.05). The strongest effect appeared at day 4 in cold season and the effect was particularly significant seen in the over 65 year-old group or in the female population. After the introduction of PM(10), SO(2) and NO(2), the concentration of PM(2.5), did not show significant effect on the number of hospital visits due to COPD on the same day (P>0.05). The effect of COPD on the fourth day showed a slight change after the lagging, and the effect was statistically significant (P<0.05). Conclusion: The increase of PM(2.5) concentration in Ningbo was related to the increase of COPD outpatient numbers. Effective prevention measures should be taken to protect the vulnerable population and to reduce the risk of COPD.

Integrative transcriptomic and genomic analysis of odorant binding proteins and chemosensory proteins in aphids.

Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play essential roles in insect chemosensory recognition. Here, we identified nine OBPs and nine CSPs from the Myzus persicae transcriptome and genome. Genomic structure analysis showed that the number and length of the introns are much higher, and this appears to be a unique feature of aphid OBP genes. Three M. persicae OBP genes (OBP3/7/8) as well as CSP1/4/6, CSP2/9 and CSP5/8 are tandem arrayed in the genome. Phylogenetic analyses of five different aphid species suggest that aphid OBPs and CSPs are conserved in single copy across all aphids (with occasional losses), indicating that each OBP and CSP class evolved from a single gene in the common ancestor of aphids without subsequent duplication. Motif pattern analysis revealed that aphid OBP and CSP motifs are highly conserved, and this could suggest the conserved functions of aphid OBPs and CSPs. Three OBPs (MperOBP6/7/10) are expressed antennae specifically, and five OBPs (MperOBP2/4/5/8/9) are expressed antennae enriched, consistent with their putative olfactory roles. M. persicae CSPs showed much broader expression profiles in nonsensory organs than OBPs. None of the nine MperCSPs were found to be antennae specific, but five of them (MperCSP1/2/4/5/6) showed higher expression levels in the legs than in other tissues. MperCSP10 mainly expressed in the antennae and legs. The broad and diverse expression patterns of M. persicae CSPs suggest their multifunctions in olfactory perception, development and other processes.

[Study on influence of air temperature on daily chronic obstructive pulmonary disease mortality in Ningbo].

Objective: To study the influence of daily air temperature on daily chronic obstructive pulmonary disease (COPD) mortality in residents in Ningbo. Methods: A time-series analysis using distributional lag non-linear model (DLNM) was conducted to estimate the attributable numbers and fraction of average air temperature on the daily COPD mortality in Ningbo from 2011 to 2016, by controlling the long-term time trend, day of week, air pollutants and other weather variables. Results: A reverse J-shape relationship was found between the average air temperature and COPD mortality, and the minimum-mortality temperature (MMT) was 25.5 ℃. The relative risks of extreme low and extreme high air temperature over lag 0-14 d were 2.767 (95%CI: 1.950-3.928) and 1.197 (95%CI: 1.021-1.404). In total, 31.62% (95%CI: 23.05%-38.89%) of COPD mortality (4 963 cases, 95%CI: 3 469-6 027) was attributable to non-MMT exposure, More attributable deaths were due to low air temperature, with a fraction of 30.41% corresponding to 4 772 deaths, compared with 1.22% and 192 deaths due to high air temperature. Low air temperature and high temperature were more likely to influence the mortality in females and those aged ≥65 years, but the influence was not significant in people aged <65 years. Conclusion: Both high and low air temperature were associated with an increased risk of COPD mortality in residents in Ningbo, especially during cold season, related measures on disease prevention should be taken to protect vulnerable population to reduce the risk of COPD mortality.

[Relationship between weather factors and heat stroke in Ningbo city].

OBJECTIVE: To explore the main effects of weather factors on heat stroke. METHODS: Data from case report on heat stroke was collected in Ningbo city during 2011 to 2014. Temperature threshold, lag effects and interaction of weather factors on heat stroke had been analyzed, using the piecewise regression model, distributed lag non-linear model, response surface model and other methods. RESULTS: RESULTS showed that temperature and humidity were more correlated with heat stroke than other weather-related factors. Through different models, daily average temperature always presented a better role in predicting the heat stroke, rather than maximum or minimum temperature. Positive association between daily average temperature and heat stroke was obvious, especially at lag 0-1 days, with its threshold as 29.1 (95% CI: 28.7-29.5) ℃ . The cumulative RR of heat stroke at 90(th) percentile of daily average temperature versus 10(th) percentile was 14.05 (95% CI: 7.23-27.31) in lag 0-1 days. The effects of daily relative humidity on heat stroke appeared nonlinear, with low humidity showing a negative effect on heat stroke and could lag for 1-4 days. However, the effect of high humidity was not significant, with the cumulative RR of low humidity and high humidity as 2.35 (95%CI: 1.27-4.33) and 0.86 (95%CI: 0.40-1.85) in lag of 0-4 days, respectively. We also noticed that there was an interactive effect of both temperature and humidity on heat stroke. Under high temperature and low humidity, the risk of heat stroke showed the highest. CONCLUSIONS: Temperature and humidity showed obvious relationship with heat stroke in Ningbo city, with the threshold temperature as 29.1 ℃. Under high temperature and low humidity, the risk of heat stroke became the highest.

[Relationship between particulate matters and cardio-cerebrovascular mortality in Ningbo: a time-series study].

OBJECTIVE: To explore the short-term effect of particulate matters with an aerodynamic diameter of less than or equal to 10 µg (PM10) and aerodynamic diameter of less than or equal to 2.5 µg (PM2.5) on cardio-cerebrovascular mortality in Ningbo city. METHODS: Daily cardio-cerebrovascular mortality data from 2011 to 2014 in Ningbo city were collected and the time series study using a semi-parametric generalized additive model were used to evaluate the relationship between the mortality of cardio-cerebrovascular disease and particulate matters after adjustment for the long-term trend of death,weather conditions, "days of the week" and other confounding factors. RESULTS: In single-pollutant model, the short-term effects of particulate matter on cardio-cerebrovascular mortality was strongest in lagged 2 days in Ningbo city, and an increase of 10 µg/m(3) in moving average concentrations (lagged 2-3 days and lagged 2-4 days) of PM2.5 and PM10 could increase the cardio-cerebrovascular mortality by 0.55% (0.23%-0.87%) and 0.53% (0.28%-0.78%), respectively. In multi-pollutant models, PM10 did remain robust after being adjusted for PM2.5 with 0.58% (0.09%-1.07%) increase in cardio-cerebrovascular mortality. The effect of PM2.5 had no statistical significantce after being adjusted for other co-pollutants. CONCLUSION: These findings suggested that the concentrations of ambient particulate matters were associated with an increased risk of daily cardio-cerebrovascular mortality in Ningbo city.

In vitro assessment of the gastrointestinal tolerance and immunomodulatory function of Bacillus methylotrophicus isolated from a traditional Korean fermented soybean food.

AIMS: This study aimed to investigate the potential of Bacillus methylotrophicus as a probiotic. METHODS AND RESULTS: A Bacillus isolate designated strain C14 was isolated from Korean traditional fermented soybean paste (doenjang). The strain was identified, and its physiological and biochemical properties were characterized. The gastrointestinal tolerance and immunomodulatory function of strain C14 were also investigated. Strain C14 was identified as B. methylotrophicus by analysis of its biochemical properties using the API 50CHB system and by phylogenetic analysis of the 16S rDNA sequence. Strain C14 showed >80% and >75% of survival for artificial gastric juices (pH 2.5 and 1% pepsin) and 0.5% (w/v) bile salt, respectively. Heat-killed B. methylotrophicus C14 inhibited the adhesion of various pathogens and enhanced the adhesion of probiotic bacteria to Caco-2 cells. The heat-killed cells also induced high levels of immune cell proliferation compared with the control and stimulated interleukin-6 and tumour necrosis factor-α production in mouse macrophages. CONCLUSIONS: Bacillus methylotrophicus C14 could be used as a probiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: Recently identified B. methylotrophicus is a new potential probiotic with high gastrointestinal tolerance.

A ring system detected around the Centaur (10199) Chariklo.

Hitherto, rings have been found exclusively around the four giant planets in the Solar System. Rings are natural laboratories in which to study dynamical processes analogous to those that take place during the formation of planetary systems and galaxies. Their presence also tells us about the origin and evolution of the body they encircle. Here we report observations of a multichord stellar occultation that revealed the presence of a ring system around (10199) Chariklo, which is a Centaur--that is, one of a class of small objects orbiting primarily between Jupiter and Neptune--with an equivalent radius of 124 ± 9 kilometres (ref. 2). There are two dense rings, with respective widths of about 7 and 3 kilometres, optical depths of 0.4 and 0.06, and orbital radii of 391 and 405 kilometres. The present orientation of the ring is consistent with an edge-on geometry in 2008, which provides a simple explanation for the dimming of the Chariklo system between 1997 and 2008, and for the gradual disappearance of ice and other absorption features in its spectrum over the same period. This implies that the rings are partly composed of water ice. They may be the remnants of a debris disk, possibly confined by embedded, kilometre-sized satellites.

The antenna-specific odorant-binding protein AlinOBP13 of the alfalfa plant bug Adelphocoris lineolatus is expressed specifically in basiconic sensilla and has high binding affinity to terpenoids.

Odorant-binding proteins (OBPs) are crucial in the olfactory pathway of insects. In the present study, the antenna-enriched OBP AlinOBP13 was investigated because of its potential contribution to the peripheral olfactory perception in the alfalfa plant bug Adelphocoris lineolatus. The results of quantitative reverse transcriptase-PCR showed that the transcript level of AlinOBP13 was higher in the adult stage than in the nymph stages. The transcript levels of AlinOBP13 in the male and female antennae significantly increased after 4 and 8 h of starvation, respectively. Fine ultrastructures of different types of chemosensilla in both female and male antennae were investigated using transmission electron microscopy and immunocytochemical labelling. The results revealed that the anti-AlinOBP13 antiserum strongly and specifically labelled short basiconic sensilla; this antiserum was restricted to the inner lumen and the cavities below the sensillum base of the sensilla. By contrast, multiporous sensilla trichodea, medium long sensilla basiconica, and aporous sensilla chaetica were not labelled. The present study is the first to report an OBP showing specific expression in the short basiconic sensilla of a member of the Hemipteran species. The results of a fluorescence displacement binding assay indicated that recombinant AlinOBP13 showed a more specific binding preference to terpenoids than to sex pheromones and other classes of chemicals. This binding ability was dramatically affected by pH; higher binding affinities were displayed at pH 10.0 than at pH 7.4 and 5.0. In addition, the results of dose-dependent electroantennogram recordings from the antennae showed that both female and male adult bugs responded to the terpenoids tested, suggesting an apparent physiological relevance of AlinOBP13 in A. lineolatus chemoreception. The results of this study suggest that AlinOBP13 functions as a specific carrier of terpenoids and provide insights into the mechanism of A. lineolatus in response to green volatiles.

High prevalence of Nova hantavirus infection in the European mole (Talpa europaea) in France.

SUMMARY: Recent discovery of genetically distinct hantaviruses in shrews and moles (order Soricomorpha, family Soricidae and Talpidae) has challenged the conventional view that rodents serve as the principal reservoir hosts. Nova virus (NVAV), previously identified in archival liver tissue of a single European mole (Talpa europaea) from Hungary, represents one of the most highly divergent hantaviruses identified to date. To ascertain the spatial distribution and genetic diversity of NVAV, we employed RT-PCR to analyse lungs from 94 moles, captured in two locations in France, during October 2012 to March 2013. NVAV was detected in more than 60% of moles at each location, suggesting efficient enzootic virus transmission and confirming that this mole species serves as the reservoir host. Although the pathogenic potential of NVAV is unknown, the widespread geographical distribution of the European mole might pose a hantavirus exposure risk for humans.

Specific involvement of two amino acid residues in cis-nerolidol binding to odorant-binding protein 5 AlinOBP5 in the alfalfa plant bug, Adelphocoris lineolatus (Goeze).

Olfaction plays an important role in insects' survival and reproduction. Odorant-binding proteins (OBPs) are considered to be one of the crucial proteins in the insect olfactory pathway. In this study, an antenna-specific OBP of the alfalfa plant bug, Adelphocoris lineolatus AlinOBP5, was expressed and purified in vitro. The binding affinities of AlinOBP5 with sex pheromone analogues of the Miridae and cotton volatiles were investigated by fluorescence competitive binding assays. The binding sites of AlinOBP5 were predicted by three-dimensional structure modelling and molecular docking, and site-directed mutagenesis. AlinOBP5 could not effectively bind with sex pheromone analogues of Miridae but showed high binding abilities with specific cotton volatiles, such as cis-nerolidol, ethyl laurate, ß-ionone, ß-caryophyllene, 2,3-dimethylbenzoic acid and (E)-farnesol. The strongest binding affinity was to cis-nerolidol, suggesting a role of AlinOBP5 in general odorant chemoreception. Based on the relatively strong binding affinity and the reported physiological activity of cis-nerolidol in other insects, we chose cis-nerolidol for further homology modelling and ligand docking. The results of molecular simulation and site-directed mutagenesis indicated that two amino acids, Lys74 and Pro121, in the protein binding pocket are the key amino acids involved in the binding of cis-nerolidol. The Lys74 residue may participate in specific recognition of ligands, and the Pro121 residue plays a crucial role in ligand binding and release by changing the binding pocket environment and stabilizing the conformation of the C-terminus of AlinOBP5.

The study of myogenin expression in denervated human skeletal muscles.

Skeletal muscle denervation eventually causes atrophy as a result of interrupted nerve conduction and the lack of nutritional factors. Myogenin is a myogenic regulatory factor that plays a key role in myoblast differentiation. Changes in myogenin expression in denervated rat skeletal muscle have been demonstrated, but myogenin expression in denervated human skeletal muscle has not been reported. Human muscle samples were analysed at different time-points post-denervation to evaluate changes in myogenin expression and their relationship with skeletal muscle atrophy. Post-denervation, myogenin mRNA levels peaked at 7 months and were 37.5 times the normal level. Expression levels then declined to 21 and 11 times the normal level at 12 and 26 months post-denervation, respectively. Prolonged denervation resulted in pathological changes characterized by decreased numbers of intact muscle fibres.

Regenerated host axons form synapses with neurons derived from neural stem cells transplanted into peripheral nerves.

It is reported that neural stem cells (NSC) can arrest denervated muscle atrophy and promote nerve regeneration when transplanted into injured peripheral nerves, and that regenerated host axons can form synapses with transplanted and differentiated NSC. In this study, F344 rat nerve segments and F344 rat NSC were transplanted into host green fluorescence protein (GFP) transgenic F344 rats. This allowed transplanted F344 rat tissue to be used as a nonluminous background for the clear visualization of regenerated host GFP axons. Regenerated host axons grew into the transplanted F344 nerve segment 2 weeks after nerve anastomosis. Immunohistochemical staining and confocal microscope analysis revealed that regenerated host axons formed synapses with NSC-derived neurons. The findings confirmed that regenerated peripheral axons form synapses with neurons in peripheral nerves, possibly forming the basis for clinical application in peripheral nerve injury.

Induction of DNA synthesis by 20-hydroxyecdysone in the prothoracic gland cells of the silkworm Bombyx mori during the last larval instar.

DNA synthesis in the prothoracic gland cells of the silkworm Bombyx mori was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the last larval instar were examined. During the first 3 days of the last larval instar, no DNA-synthesizing cells were detected. On day 4, the number of DNA-synthesizing cells increased and peaked on day 7. When larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves throughout the last larval instar, the number of DNA-synthesizing cells dramatically increased 2 days after 20-hydroxyecdysone treatment and reached a high level on day 5. The mechanism by which 20-hydroxyecdysone treatment induces DNA synthesis of prothoracic gland cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. We conclude that 20-hydroxyecdysone may exert its growth-promoting action indirectly.

[Study on the expression of pig zona pellucida-3 beta excluding N-terminus signal peptide and C-terminus transmembrance-like domain in Escherichia coli].

Pig oocytes are surrounded by an acellular translucent envelope-zona pellucida(pZP), which is comprised of three biochemically and immunologically distinct glycoproteins(pZP1, pZP3 alpha and pZP3 beta). Due to the cross-reactivity of anti-pZP3 beta antibodies with human ZP in vitro, pZP3 beta has been considered as potential one of immunogens for developing human contraceptive vaccine. In order to express pZP3 beta directly in E. coli; we amplified the core fragment of pZP3 beta cDNA by PCR that was deleted 5'- and 3'-terminal sequences of coding for signal peptide and transmembrane-like domain. The DNA sequenced EcoRI and SalI restriced core fragment was cloned in a frame downstream of PRPL promoter in the pBV221 vector. SDS-PAGE analysis showed that pZP3 beta protein was expressed especially in E. coli after thermal induction, which the expression band displayed the molecular weight of about 38 kD matching with its deduced molecular weight(36.5 kD). In addition, the specially expressed protein band on SDS-PAGE gel showed specific immunological reaction with anti-pig ZP IgGs of rabbit in Western blot.

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori.

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.