Molecular cloning and transcription expression of 3-dehydroecdysone 3α-reductase (3de 3α-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, and reproduction etc. The decreased ecdysteroid in titre results from enhanced ecdysteroid inactivation reactions including the formation of 3-epiecdyson under ecdysone oxidase and 3-dehydroecdysone 3α-reductase (3DE 3α-reductase). In this paper, we cloned and characterized 3-dehydroecdysone 3α-reductase (3DE 3α-reductase) in different tissues and developing stage of the silkworm, Bombyx mori L. The B. mori 3DE 3α-reductase cDNA contains an ORF 783 bp and the deduced protein sequence containing 260 amino acid residues. Analysis showed the deduced 3DE 3α-reductase belongs to SDR family, which has the NAD(P)-binding domain. Using the Escherichia coli, a high level expression of a fusion polypeptide band of approx. 33 kDa was observed. High transcription of 3DE 3α-reductase was mainly presented in the midgut and hemolymph in the third day of fifth instar larvae in silkworm. The expression of 3DE 3α-reductase at different stages of larval showed that the activity in the early instar was high, and then reduced in late instar. This is parallel to the changes of molting hormone titer in larval. 3DE 3α-reductase is key enzyme in inactivation path of ecdysteroid. The data elucidate the regulation of 3DE 3α-reductase in ecdyteroid titer of its targeting organs and the relationship between the enzyme and metamorphosis.

Pancreatic neuroendocrine tumor detected by technetium-99m methoxy-2-isobutylisonitrile single photon emission computed tomography/computed tomography: A case report.

BACKGROUND: Pancreatic neuroendocrine tumors (NETs) account for about 1%-2% of pancreatic tumors and about 8% of all NETs. Computed tomography (CT), magnetic resonance imaging, and endoscopic ultrasound are common imaging modalities for the diagnosis of pancreatic NETs. Furthermore, somatostatin receptor imaging is of great value for diagnosing pancreatic NETs. Herein, we report the efficacy of technetium-99m methoxy-2-isobutylisonitrile (99mTc-MIBI) single photon emission CT (SPECT)/CT for detecting pancreatic NETs. CASE SUMMARY: A 57-year-old woman presented to our hospital with a 1-d history of persistent upper abdominal distending pain. The distending pain in the upper abdomen was aggravated after eating, with nausea and retching. Routine blood test results showed a high neutrophil percentage, low leukomonocyte and monocyte percentages, and low leukomonocyte and eosinophil counts. Amylase, liver and kidney function, and tumor markers alpha-fetoprotein, carcinoembryonic antigen, and cancer antigen (CA) 125, CA72-4, CA19-9, and CA153 were normal. Abdominal CT showed a mass, with multiple calcifications between the pancreas and the spleen. The boundary between the mass and the pancreas and spleen was poorly defined. Contrast-enhanced CT revealed that the upper abdominal mass was unevenly and gradually enhanced. 99mTc-MIBI SPECT/CT revealed that a focal radioactive concentration, with mild radioactive concentration extending into the upper abdominal mass, was present at the pancreatic body and tail. The 99mTc-MIBI SPECT/CT manifestations were consistent with the final pathological diagnosis of pancreatic NET. CONCLUSION: 99mTc-MIBI SPECT/CT appears to be a valuable tool for detecting pancreatic NETs.

Expression of Trichoderma viride endoglucanase III in the larvae of silkworm, Bombyx mori L. and characteristic analysis of the recombinant protein.

Endoglucanase is a part of cellulase which hydrolyzes cellulose into glucose. In this study, we cloned endoglucanase III (EG III) gene from Trichoderma viride strain AS 3.3711 using a PCR-based exon splicing method, and expressed EG III recombinant protein in both silkworm BmN cell line and silkworm larvae with an improved Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the chiA and v-cath genes of Bombyx mori nucleopolyhedrovirus (BmNPV). The result showed that around 45 kDa protein was visualized in BmN cells at 48 h after the second generation recombinant mBacmid/BmNPV/EG III baculovirus infection. The enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 8.0 and temperature 50°C, and increased 20.94 and 19.13% compared with that from blank mBacmid/BmNPV baculoviruses infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 9.0 and at temperature range from 40 to 60°C. It provided a possibility to generate transgenic silkworms expressing bio-active cellulase, which can catabolize dietary fibers more efficiently, and it might be of great significance for sericulture industry.

Cloning and characterization of the Bombyx mori ecdysone oxidase.

The physiological titer of molting hormones in insects depends on relative activities of synthesis and degradation pathways. Ecdysone oxidase (EO) is a key enzyme in the inactivation of ecdysteroid. However, there are only a few reports on ecdysteroid inactivation and its enzymes in silkworm. In this study, we cloned and characterized the Bombyx mori EO (BmEO). The BmEO cDNA contains an ORF of 1,695 bp and the deduced protein sequence contains 564 amino acid residues. The deduced protein sequence contains two functional domains of glucose-methanol-choline oxidoreductase in N-terminal and C-terminal. Comparing the expression levels of BmEO in different tissues, high transcription was mainly present in hemocytes. Reduced expression of this enzyme is expected to lead to pathological accumulation of ecdysone in the hemolymph of silkworm larvae or pupae. Our data show that RNA inference of BmEO transcripts resulted in the accumulation of ecdysteroid and death of larvae or pupae. We infer that EO is a crucial element in the physiology of insect development.

Cloning and expression pattern of 3-dehydroecdysone 3ß-reductase (3DE 3ß-reductase) from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, reproduction, etc. Ecdysone was inactivated to 3-dehydroecdysone (3DE) under ecdysone oxidase (EO), and followed by NAD(P)H-dependent irreversible reduction to 3-epiecdysteroid under 3DE 3a-reductase. On the other hand, 3-dehydroecdysone undergoes reversible reduction to ecdysone by 3DE 3ß-reductase in the hemolymph. In this article, we cloned and characterized 3-dehydroecdysone 3ß-reductase (3DE 3ß-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L. The B. mori 3DE 3ß-reductase cDNA contains an ORF 972 bp and the deduced protein sequence containing 323 amino acid residues. Analysis showed that the deduced 3DE 3ß-reductase belongs to the aldo-keto reductase (AKR) superfamily, which has the NAD(P)-binding domain, indicating that the function of 3DE 3ß-reductase depends on the existence of NAD(P)H. Using Escherichia coli, a high level expression of a fusion polypeptide band of approx. 40 kDa was observed. The high transcription of 3DE 3ß-reductase was mainly observed in the genitalia and fatty bodies in the third day of the fifth-instar larvae, followed next in the head, epidermis, and hemocytes. The expression of 3DE 3ß-reductase in the early of every instar was lower than that in the late of instar. When the titer of 3DE is low, higher expression of 3DE 3ß-reductase is necessary to maintain the ecdysone titer in body through converting 3DE to ecdysone, while the 3DE titer is high, the expression of 3DE 3ß-reductase showed feedback inhibition.

Demonstration of protein absorption in the intestinal epithelium of fish and mice by laser scanning confocal microscopy.

Selective permeability for small proteins and oligopeptides occurs in the intestinal epithelium of many animal species and humans. Whole proteins are sometimes endocytosed and undergo partial hydrolysis in intestinal epithelial cells with the probable release of essential oligopeptides into the bloodstream. Increased permeability to certain proteins can cause asthma and other metabolic disorders. Permeable proteins have also been successfully used to deliver vaccines or drugs via oral consumption. Protein absorption has been inferred in many cases and demonstrated in some cases by histochemical, tracer, and analytical techniques. However, the nature and importance of protein absorption remains largely unknown. Here, we demonstrate the movement of two lumenal proteins (GFP: 26 kDa and OFP: 23 kDa) across the intestinal epithelium of fish and mice using laser scanning confocal microscopy. The results provide evidence that small proteins can be taken up intact by intestinal epithelial cells, even though large proteins are digested to single amino acids or protein fragments before they are absorbed. Our results suggest that it is possible to orally administer some small proteinous medicines for therapeutic purposes.

Cloning and expression of a cellulase gene in the silkworm, Bombyx mori by improved Bac-to-Bac/BmNPV baculovirus expression system.

Cellulases catalyze the hydrolysis of cellulose which are mainly three types: endoglucanases, cellobiohydrolases and ß-glucosidases. It can be used in converting cellulosic biomass to glucose that can be used in different applications such as production of fuel ethanol, animal feed, waste water treatment and in brewing industry. In this paper, we cloned a 1380-bp endoglucanase I (EG I) gene from mycelium of filamentous fungus Trichoderma viride strain AS 3.3711 using PCR-based exon splicing methods, and expressed the recombinant EG I mature peptide protein in both silkworm BmN cell line and silkworm larvae with a newly established Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV). An around 49-kDa protein was visualized after mBacmid/BmNPV/EG I infection, and the maximum expression in silkworm larvae was at 84 h post-infection. The ANOVA showed that the enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 7.0 and temperature 50°C. It was stable at pH range from 5.0 to 10.0 and at temperature range from 50 to 60°C, and increased 24.71 and 22.84% compared with that from wild baculoviruses infected silkworms and normal silkworms, respectively. The availability of large quantities of EG I that the silkworm provides maybe greatly facilitate the future research and the potential application in industries.

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L.

The peritrophic membrane (PM) is a semi-permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin-binding proteins (BmPM-P43 and BmPM-P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C-terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM-P43 and BmPM-P41. The chitin-binding activity analysis showed that the BmPM-P43 and BmPM-P41 could bind to chitin strongly. It is concluded that BmPM-P43 and BmPM-P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin-binding protein family.

Screening and identification of new isolate: thermostable Escherichia coli with novel thermoalkalotolerant cellulases.

A cellulase-producing bacterium strain was isolated from soil that produced novel thermoalkalotolerant cellulases after growth on CMC-Na agar screening plate at 37 degrees C. It was identified as Escherichia coli using the method of 16S rRNA and intergenic spacer gene analysis combined with morphological, physiological, and biochemical tests. Three major components of the cellulases [carboxymethyl cellulase (CMCase), filter paper cellulase, and beta-glucosidase] were produced with maximal activities (0.23, 0.08, and 0.15 U/ml) and maximum specific activities 4.13, 0.56, and 0.50 U/mg protein after 72, 96, and 120 h growth, respectively. Maximum CMCase activity was measured at 50 degrees C and pH 6.0, respectively, and it also retained more than 60% of its maximal activity for at least 20 min at 50-70 degrees C and 10 min at 80 degrees C, respectively, and retained approximately 50% of its maximal activity after incubating at 90 degrees C for 10 min. The enzyme could be applied in bioconversion of lignocellulosic agricultural wastes.

[MRI diagnosis of benign sacrococcygeal teratomas of the infants].

OBJECTIVE: To investigate the value of MR imaging in the diagnosis of benign sacrococcygeal teratomas of the infants. METHODS: MR imaging of benign sacrococcygeal teratomas in 6 cases proved by surgery and pathology was retrospectively reviewed. In all patients, a fast imaging sequence, fast spin echo sequence was employed, together with short time inversion recovery sequence and contrast enhancement scanning by intravenous injection of Gd-DTPA. RESULTS: There were 6 benign sacrococcygeal teratomas, which were heterogeneous masses and arose from the distal sacrococcygeal region in the pelvis. The MR imaging appearances of the benign sacrococcygeal teratomas were characteristic, T1- and T2-weighted images demonstrated a large mass containing round, well-defined areas of varying signal intensity representing its cystic, solid, and sometimes fat, calcification within the lesions. CONCLUSION: MR imaging provides definitive information of benign sacrococcygeal teratomas and clearly shows both extra-and intra-pelvic components, and even better anatomic details, which facilitates the surgical planning adequately.

Effect of a multi-level education intervention model on knowledge and attitudes of accidental injuries in rural children in Zunyi, Southwest China.

OBJECTIVE: To explore the effect of a school-family-individual (SFI) multi-level education intervention model on knowledge and attitudes about accidental injuries among school-aged children to improve injury prevention strategies and reduce the incidence of pediatric injuries. METHODS: The random sample of rural school-aged children were recruited by using a multistage, stratified, cluster sampling method in Zunyi, Southwest China from 2012 to 2014, and 2342 children were randomly divided into intervention and control groups. Then children answered a baseline survey to collect knowledge and attitude scores (KAS) of accidental injuries. In the intervention group, children, their parents/guardians and the school received a SFI multi-level education intervention, which included a children's injury-prevention poster at schools, an open letter about security instruction for parents/guardians and multiple-media health education (Microsoft PowerPoint lectures, videos, handbooks, etc.) to children. Children in the control group were given only handbook education. After 16 months, children answered a follow-up survey to collect data on accidental injury types and accidental injury-related KAS for comparing the intervention and control groups and baseline and follow-up data. RESULTS: The distribution of gender was not significantly different while age was different between the baseline and follow-up survey. At baseline, the mean KAS was lower for the intervention than control group (15.37 ± 3.40 and 18.35 ± 5.01; p < 0.001). At follow-up, the mean KAS was higher for the intervention than control group (21.16 ± 3.05 and 20.02 ± 3.40; p < 0.001). The increase in KAS in the intervention and control groups was significant (p < 0.001; KAS: 5.79 vs. 1.67) and suggested that children's injury-related KAS improved in the intervention group. Moreover, the KAS between the groups differed for most subtypes of incidental injuries (based on International Classification of Diseases 10, ICD-10) (p < 0.05). Before intervention, 350 children had reported their accident injury episodes, while after intervention 237 children had reported their accidental injury episodes in the follow-up survey. CONCLUSIONS: SFI multi-level education intervention could significantly increase KAS for accidental injuries, which should improve children's prevention-related knowledge and attitudes about such injuries. It should help children change their risk behaviors and reduce the incidence of accidental injuries. Our results highlight a new intervention model of injury prevention among school-aged children.

Escalated hyperfractionated accelerated radiation therapy for locally advanced non-small cell lung cancer: a clinical phase II trial.

BACKGROUND AND PURPOSE: To evaluate the feasibility, toxicity and the efficacy for locally advanced non-small cell lung cancer (NSCLC) treated with escalated hyperfractionated accelerated radiation therapy (EHART) combined with chemotherapy. PATIENTS AND METHODS: The EHART consisted of irradiation delivered twice per day with >6-h interval and five treatment days per week. In the first and second weeks, 1.2 Gy/fraction b.i.d, was given, and then 1. /fraction b.i.d in the third week; 1.4 Gy/fraction b.i.d in the fourth week; and 1. /fraction b.i.d in the fifth week, respectively. The total tumor dose delivered was 66 Gy/50 fractions/5 weeks. All patients received neoadjuvant and adjuvant chemotherapy. The chemotherapeutic regimen used was either MVP (mitomycin C, vindesine, cis-platinum), or EP (etoposide and cis-platinum). RESULTS: From February 1997 to February 1999, 73 eligible patients were registered. All were in stage IIIb with median age of 60 years (33-70). Of the 73 patients, 12 cases were withdrawn from the study due to Grade (Gr) III acute complications, distant metastases, or intercurrent diseases. Sixty-one patients completed the combined treatment as planned. A median of 4 cycles of chemotherapy (1-7) was administered and 66 Gy/50 fractions/36 days was delivered finally. The most common acute complication was radiation esophagitis, which occurred in 56 cases (77%), with Gr III in 11 cases (15%). Twenty-nine patients (40%) had acute pulmonary toxicity; with Gr III in 6 cases (8%). The median survival time was 13 months for the entire group. The 1-year and 2-year survival rates were 51 and 10%, respectively. Of the 61 patients who finished EHART, 34 were found to have locoregional progression. Thirty-two patients failed inside radiation fields, and 2 patients, outside radiation fields. The 1-year and 2-year locoregional progression-free rates were 71 and 34%, respectively. The 1-year and 2-year distant metastasis rates were 57 and 84%, respectively. CONCLUSIONS: EHART combined with chemotherapy could be tolerated by most of the stage IIIb NSCLC patients with acceptable complications. Locoregional control was improved, but the long survival was not prolonged significantly predominantly due to distant metastases.

Evaluation of regional bulbar redness using an image-based objective method.

AIM: To develop an image-based objective method to precisely evaluate regional ocular bulbar injection. METHODS: Six healthy adult volunteers were photographed in four orientations (superior, inferior, nasal and temporal sides) with and without stimulating eye drops. Six line segments (covering 30°) were drawn 4mm away from the limbus on each image using ImageJ software. The graph peaks, which were derived from the areas under the line segments and corresponded to the cross-sectional grey-level of the vessels, were analyzed to obtain peak area, peak height/width (PH/PW), and peak numbers. Different-sized areas were selected to calculate the pixels based on the edge-detection algorithm. Also, conjunctival and superficial scleral vessels were analyzed separately. RESULTS: This method had a smaller coefficient of variation, especially for PH/PW, in all four orientations. Hyperaemia parameters changed the least after challenging in the superior region. Moreover, 95% of the PH/PW ratios were greater than 0.87 in conjunctival vessels and less than 1.00 in superficial scleral vessels. PH/PW significantly increased in conjunctival vessels and changed less in superficial scleral vessels. CONCLUSION: A new method of objectively assessing bulbar injection based on ocular surface images was developed. This method can be used to quantify ocular regional injection and to distinguish the superficial scleral and conjunctival vessels.

Heterologous expression characteristics of Trichoderma viride endoglucanase V in the silkworm, Bombyx mori L.

Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases, endoglucanases, and ß-1,4-glucosidase. In this study, we used an improved Bac-to-Bac/BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in silkworm larvae. The result showed that an around 36-kDa protein was visualized in BmN cells at 48 h after the second-generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The crude enzyme extract from the recombinant baculoviruses-infected silkworms exhibited a significant maximum activity at the environmental condition of pH 5.0 and a temperature of 50 °C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV baculovirus-infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 10.0 and at temperature range from 40 to 60 °C. The availability of large quantities of EG V that the silkworm provides might greatly facilitate the future research and the potential application in industries.

Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet.

Cellulase-producing fungi Trichoderma viride were cultured and fermented on the solid-state wheat bran fermentation medium. The characteristics of its carboxymethyl cellulase (CMCase) in the condition of this solid-state fermentation were evaluated, and the optimum culture time, optimum pH and optimum temperature for CMCase activity of T. viride fermented in this solid state were 60h, 5.0 and 50 degrees C, respectively. Carboxymethyl cellulose sodium (CMC-Na) and Congo red were used to screen the strains that had stronger ability to produce enzymes. After the compound mutagenesis by microwave and ultraviolet, seven mutant strains (M-B1-M-B7) were selected and their CMCase activities were assayed. Five of them (M-B1, M-B2, M-B3, M-B5 and M-B7) had significantly stronger ability to produce enzymes than the normal wild type, and they were also very stable for a long period up to 9 generations to produce cellulase. Molecular studies showed that there were some base mutations in endoglucanase I (EG I) genes of mutants M-B1, M-B2, M-B3 and M-B5, but no change in M-B7, suggesting that some amino mutations in EG I proteins caused by base mutations could lead to enhanced cellulase production.