A serine protease homologue Bombyx mori scarface induces a short and fat body shape in silkworm.

Body shape is one of the most prominent and basic characteristics of any organism. In insects, abundant variations in body shape can be observed both within and amongst species. However, the molecular mechanism underlying body shape fine-tuning is very complex and has been largely unknown until now. In the silkworm Bombyx mori, the tubby (tub) mutant has an abnormal short fat body shape and the abdomen of tub larvae expands to form a fusiform body shape. Morphological investigation revealed that the body length was shorter and the body width was wider than that of the Dazao strain. Thus, this mutant is a good model for studying the molecular mechanisms of body shape fine-tuning. Using positional cloning, we identified a gene encoding the serine protease homologue, B. mori scarface (Bmscarface), which is associated with the tub phenotype. Sequence analysis revealed a specific 312-bp deletion from an exon of Bmscarface in the tub strain. In addition, recombination was not observed between the tub and Bmscarface loci. Moreover, RNA interference of Bmscarface resulted in the tub-like phenotype. These results indicate that Bmscarface is responsible for the tub mutant phenotype. This is the first study to report that mutation of a serine protease homologue can induce an abnormal body shape in insects.

Ultrabithorax and abdominal-A specify the abdominal appendage in a dosage-dependent manner in silkworm, Bombyx mori.

In insects, there is a considerable diversity in leg distribution on the body, including number, segmental arrangement, morphological identity and consequent function, but the genetic basis for these differences is not well understood. Here by positional cloning, we showed that a ~355 kb region, including Bombyx mori Ultrabithorax (BmUbx) and abdominal-A (Bmabd-A), was responsible for the silkworm mutant Kh-extra-crescents-like (EKh-l) that displayed additional thoracic limb-like legs on the first abdominal segment (A1) and occasionally on the second abdominal segment (A2). We found that BmUbx gene was downregulated at both messenger RNA level and protein level in EKh-l embryo, while its expression domain in the EKh-l embryo was almost the same as that in the wild type. Whereas Bmabd-A was upregulated at both levels and was ectopically overexpressed on the supernumerary leg-bearing segments in EKh-l. Compared with the previously reported Ecs-l mutant in which increased expression of both BmUbx and Bmabd-A gave rise to ectopic proleg-like appendages on the same segments, we propose that overexpressed Bmabd-A gene is capable to promote the outgrowth of extra leg appendages on A1 and A2 segments, whereas BmUbx gene is required to specify accurate morphologies of the ectopic legs in a dosage-dependent manner in silkworm. These results provide insights into how these hox genes regulate the leg morphologic diversity on the same segments.

Morphological characterization and molecular mapping of an irradiation-induced Speckled mutant in the silkworm, Bombyx mori.

Speckled (Spc), an X-ray-induced lethal mutant of Bombyx mori, exhibits a mosaic dark-brown-spotted larval epidermis in both sexes and egg-laying problems only in females. Here, we report the morphological characterization and molecular mapping of the Spc mutant. Morphological investigations revealed that the epidermal ultrastructure of the small, dark-brown spots was more dense than that of the white regions in both Spc/+ mutants and wild type, and that the lethality of the Spc/Spc mutants occurred during early embryogenesis. Furthermore, the ovarioles and ovipositor were disconnected in approximately 85.5% of Spc/+ females, a further 2.5% had a connection between the ovarioles and ovipositor that was too narrow to lay eggs. The remaining females showed a normal connection similar to that of the wild type. We successfully narrowed down the location of the Spc mutation to a region on chromosome 4 that was ∼1041 kb long. Gene-prediction analysis identified 25 candidate genes in this region. Chromosome structure analysis indicated that a ∼305 kb deletion was included in the mapping region. Temporal and spatial reverse transcription PCR (RT-PCR) analysis showed that several genes in the mapped region are associated with the Spc mutant. Although the genes responsible for the Spc mutation were not definitively identified, our results further the current understanding of the complex mechanism underlying the multiple morphological defects in Spc mutants.

Marker-assisted selection in breeding silkworm strains with high tolerance to fluoride, scaleless wings, and high silk production.

In the silkworm (Bombyx mori), tolerance to fluoride and scaleless wings are controlled by the dominant gene Dtf (dominant tolerance to fluoride) and recessive gene nlw (no Lepidoptera wings), respectively, and these genes have been mapped by using simple sequence repeat and sequence tag site markers. Marker-assisted evaluation and selection of silkworms with fluoride tolerance and scaleless wings were used for predicting fluoride resistance and scaleless wings in backcrossed animals. A silkworm strain was bred using this method, and its economic characteristics were found to be similar to those of commercial silkworms. These methods will therefore be useful for silkworm breeding programs and in screening for two or more characteristics of interest for segregating populations.

Fine mapping of a supernumerary proleg mutant (E(Cs) -l) and comparative expression analysis of the abdominal-A gene in silkworm, Bombyx mori.

Patterning and phenotypic variations of appendages in insects provide important clues on developmental genetics. In the silkworm Bombyx mori, morphological variations associated with the E complex, an analogue of the Drosophila melanogaster bithorax complex, mainly determine the shape and number of prolegs on abdominal segments. Here, we report the identification and characterization of the allele responsible for the supernumerary crescents and legs-like (E(Cs) -l) mutant, a model derived from spontaneous mutation of the E complex, with supernumerary legs and extra crescents. Fine mapping with 1605 individuals revealed a ∼68 kb sequence in the upstream intergenic region of B. mori abdominal-A (Bmabd-A) clustered with the E(Cs) -l locus. Quantitative real-time PCR (qRT-PCR) and Western blotting analyses disclosed a marked increase in Bmabd-A expression in the E(Cs) -l mutant at both the transcriptional and translational levels, compared to wild-type Dazao. Furthermore, we observed ectopic expression of the Bmabd-A protein in the second abdominal segment (A2) of the E(Cs) -l mutant. Our results collectively suggest that the 68 kb region contains important regulatory elements of the Bmabd-A gene, and provide evidence that the gene is required for limb development in abdominal segments in the silkworm.

Antennapedia is involved in the development of thoracic legs and segmentation in the silkworm, Bombyx mori.

Homeotic genes, which are associated closely with body patterning of various species, specify segment identity. The Wedge eye-spot (Wes) is a new homeotic mutant located on the sixth linkage group. Homozygous Wes/Wes embryos are lethal and display a pair of antenna-like appendages under the mouthparts as well as fused thoracic segments. These mutants also exhibit a narrower eye-spot at the larval stage compared with the wild type. By positional cloning, we identified the candidate gene of the Wes locus, Bombyx mori Antennapedia (BmAntp). Two BmAntp transcripts were identified in the homozygote of the Wes mutant, including a normal form and an abnormal form with a 1570-bp insertion. Our data showed that the insertion element was a long interspersed nuclear element (LINE)-like transposon that destroyed the original open reading frame of BmAntp. Quantitative RT-PCR analysis showed that the expression levels of normal BmAntp transcripts were increased markedly in the Wes heterozygous larvae compared with the wild type. Furthermore, we performed RNAi of BmAntp and observed fused thoracic segments and defective thoracic legs in the developing embryos. Our results indicated that BmAntp is responsible for the Wes mutant and has an important role in determining the proper development of the thoracic segments. Our identification of a homeotic mutation in the silkworm is an important contribution to our understanding of the regulation of Hox genes at different levels of expression.

Effect of Ageratina adenophora on hepatic and pulmonic pathological lesions in horses.

The effect of Ageratina adenophora on pathological characteristics of the liver and lungs as well as serum biochemical parameters in horses were investigated. Ten horses without ingestion history of Ageratina adenophora were classified into the control group, and 10 poisoned but survived horses with 3 months ingestion history were set as the case group. Results showed that serum AST, ALT, ALP, magnesium and phosphorus were elevated significantly, while creatinine was decreased remarkably. Hematoxylin and eosin staining of liver tissues showed diffuse swelling or destruction of hepatocytes, narrowing or atrophy of the hepatic sinusoids, and little lymphocytic infiltration; lung tissues presented destroyed alveoli and inflammatory cell infiltration.

The protective effects of Berberine and Hesperidin on inflammatory factor-stimulating cardiac fibroblasts.

OBJECTIVE: The previous work has shown that Berberine and Hesperidin have beneficial effects on cardiovascular diseases. However, the underlying mechanisms remain unknown. This study aimed to investigate the effect of Berberine and Hesperidin on inflammatory cytokine secretion, proliferation, differentiation, and collagen synthesis of cardiac fibroblasts stimulated by the transforming growth factor-ß1 (TGF-ß1), and the potential of these drugs to regulate the Notch1 signaling pathway. PATIENTS AND METHODS: Neonatal rat primary cardiac fibroblasts were stimulated with 5 ng/mL TGF-ß1 as model (TGF) group. In the Berberine (TGF+B) group cells were given TGF-ß1, along with 1.25/2.5/5/10 mg/L Berberine, while the Hesperidin (TGF+H) group was treated with TGF-ß1 and 12.5/25/50/100 µmmol/L Hesperidin. Cellular proliferation, differentiation, and collagen synthesis were evaluated. The role of the Notch1 signaling pathway in the protective effects of Berberine and Hesperidin was analyzed by using γ-secretase inhibitor (DAPT) to block the Notch1 pathway. RESULTS: 5/10 mg/L Berberine intervention could noticeably decrease both TGF-ß1 and IL-1ß levels, 25/50/100 µmol/L Hesperidin could reduce IL-1ß secretion from TGF-ß1 stimulated cardiac fibroblasts. Both Berberine and Hesperidin decreased the expression of α-SMA and cell viability in a concentration-dependent manner; however, the apoptosis of cardiac fibroblasts was not influenced. 10 mg/L Berberine or at least 50 µmol/L Hesperidin could noticeably decrease MMP-1 expression, and at least 5 mg/L Berberine or 100 µmol/L Hesperidin could markedly reduce MMP-9 expression. Using DAPT to block Notch1 signaling could reverse the protective effects of Berberine and Hesperidin. CONCLUSIONS: Berberine and Hesperidin can reduce the secretion of inflammatory cytokines, differentiation, and proliferation, and increase the collagen synthesis of cardiac fibroblasts stimulated by TGF-ß1 via the Notch1 signaling pathway.

Identification and expression of the achaete-scute complex in the silkworm, Bombyx mori.

Recently, the study of achaete-scute (AS-C) homologues has contributed enormously to understanding of gene duplication and function evolution, particularly in Diptera. We identified four AS-C homologue genes in the silkworm, Bombyx mori, referred to as BmASH, BmASH2, BmASH3, and Bmase. The complex displayed tandem array structure in the genome. Analysis of spatial expression profiles showed that they all were expressed in obviously higher levels in wing disc than in other tissues, suggesting that they might play important roles in the development of the wing. Furthermore, we found that their expression profiles in the wing discs were mostly correlated with the development of the scales, especially the BmASH gene. RNA interference results further indicated that BmASH was necessary for scale formation in silkworm wing.

A comprehensive neurobehavioral and neurophysiological study for low level lead-exposed workers.

UNLABELLED: A comprehensive neurobehavioral and neurophysiological study was performed to evaluate the adverse effect of low level lead-exposure, and to compare the sensibility, easiness of the test methods utilized. The tests were: WHO recommended Neurobehavioral Core Test Battery (NCTB), Autonomic Nouvers System Function (ANS) Test Battery, Brain Electricity Active Mapping (BEAM), and Nerve Conduction Velocity. 44 lead-exposed workers were selected, with 34 age, education degree, family economic level, smoking and drinking matched referents. RESULT: The mean blood lead concentration of lead-exposed workers was 1.3870 mumol/L, whereas that of referents was 0.6080 mumol/L, the difference was very significant. The negative Profile of Mood State (POMS) score of lead-expose workers was higher than that of referents, whereas the positive POMS score of the referents was higher than that of lead-exposed group, with a covariance analysis. The lead-exposure affected some NCTB test items, such as simple reaction time (SRT), digital symbol (DSY), correct dots (PAC) and total dots (PA). The heart-rate response to Valsalva manoeuvre (HR-V), heart-rate response to deep breathing (HR-DB), and blood-pressure response to immediate standing (BP-IS) were lowered in lead-exposed workers significantly. Some abnormal brain electric waves (dominant beta frequency, semetry-diffuse abnormal and non semetry-diffuse abnormal wave distribution, dominant low wave amplitude) appeared in lead-exposed workers. Left ulnar nerve maximal conduction velocity was significantly lowered in lead-exposed group. CONCLUSION: The NCTB (including POMS), and ANS function test should be the regular screening battery for low level lead-exposed workers. The threshold blood lead concentration for health surveillance should be 30 micrograms/dL, or 1.4 mumol/L.