Cyclic AMP response element binding protein (CREB) participates in the heat-inducible expression of humanhsp90β gene

Human heat shock protein 90b gene ( hsp90b ) is a constitutively expressed heat shock gene existing in most of cell types tested that can be further induced by heat shock. Chloramphenical acetyl transferase (CAT) reporter plasmids driven by different regulatory fragments of hsp90b gene were constructed and transfected into Jurkat cells to explore the role of a cAMP response element (CRE) in the upstream of the gene. Results show that, in comparison with the wild type construct, a severe reduction (~2/3) in the increased folds of promoter activity induced by heat shock at 42℃ for 1 h was observed in a construct with CRE-containing fragment (-173/-91bp) deleted. Electrophoretic mobility shift assays (EMSA) showed that phosphorylated CRE-binding protein (CREB) in the nuclear extract of heat shocked Jurkat cells is specifically bound to the fragment. Additionally, both of the phosphorylation on CREB and the activity of protein kinase A (PKA) were found in Jurkat cells to be enhanced with extending time of heat shock treatment. Our results indicate that in addition to the intronic HSE/HSF pathway, phosphorylated CREB also participates in the heat shock induced expression of human hsp90b gene via its interaction with CRE which may be regulated by PKA-sig- naling pathway.     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS) alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin α from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3′ end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

Immunolocalization of aromatase, estrogen and estrogen receptor α and β in the epithelium of digestive tract and enteric neurons of amphioxus (Branchiostoma belcheri )

Immunohistochemical localization of aromatase, estrogen and estrogen receptor in the digestive tract and enteric neurons of amphioxus is investigated. It was found that immunoreactive proteins of aromatase, estrogen and ER-α and β are expressed in hepatic diverticulum, epithelial cells of anterior and posterior region of midgut, as well as in enteric neurons, while hindgut showed immunonegative. The results suggest that digestive tract of amphioxus may be able to synthesize estrogen and possess endocrine function, like rat gastric epithelium and enteric neurons in mammals. The present study provides authentic morphological evidence for explaining the action mechanism of estrogen in regulating the digestive function of gut and the functional evolution of estrogen, which has important theoretical significance in amphioxus.     

Molecular cloning and expression of interleukin Ibeta (IL-1β) from red seabream (Pagrus major)

The interleukin 1β (IL-1β) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1β sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1β (RS IL-1β). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1β genes. The RS IL-1β had the highest homology with piscine IL-1β according to phylogenetic tree analysis.     

Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles ste- phensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I di- gestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively con- served in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.     

The 5′ untranslated region of fruA mRNA in Myxococcus xanthus positively regulates the expression of its own gene

Studyingthemolecularmechanismsgoverningmorphogenesisofmulticellularstructureandcelldifferen-tiationhasbeenoneofthecentralthemesinlifesciences.M.xanthus,aGram-negativebacterium,isoneofthesim-plestorganismsthatundergoaprogramofmulticellulardevelopment.Inresponsetonutrientstarvation,approxi-mately100000cellsmovetowardanaggregationcentertoformafruitingbody.Withinthenascentfruitingbody,rod-shapedcellsgraduallyshortenanddifferentiateintoroundmyxospores.Sporesgerminatetoinitiatevegetativegrowthifnut…     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS} alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin a from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3' end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

NO and H2O2 induced by Verticillium dahliae toxins and its influence on the expression of GST gene in cotton suspension cells

Nitric oxide （NO） and hydrogen peroxide （H2O2） have been shown to be important signaling molecules that participate in the regulation of several physiological processes. In particular, they have significant role in plant resistance to pathogens by contributing to induction defense genes. Here, whether NO and H2O2 participate in the resistance responses against Verticillium dahliae toxins （VD-toxins） and their effects on the expression of GSTgene are studied. The results reveal that NO and H2O2 are produced as part of a complex network of signals that respond to VD-toxins and may converge to function both synergistically and independently by inducing resistant responses. GSTgene is potentially involved in the resistance mechanism in the cotton suspension cells. NO induces the expression of GSTgene independently of H2O2. H2O2 may be a more potent signal in the resistance responses against VD-toxins.     

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F₀ mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

GABAA(δ) receptor hypofunction in the amygdala-hippocampal circuit underlies stress-induced anxiety

Dysregulated GABAergic inhibition in the amygdala has long been implicated in stress-related neuropsy-chiatric disorders.However,the molecular and circuit mecha...     

Cloning， expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except ATP. Here we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned using yeast two-hybrid approach. A tobacco NDPK gene was obtained and designated as NtNDPK1. NtNDPK1 is 704 bp in length and encodes a putative 16.2 kD protein of 148 amino acids. Phylogenic analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type I (NDPK1). RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 expression in roots, stems, leaves and buds. And expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl, but not significantly affected by Paraquat, wounding and low temperature (4℃) treatments, indicating that NtNDPK1 may play a certain role in response to abiotic stress. In vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphorylation activity.     

Cloning， expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except ATP.Here we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned us- ing yeast two-hybrid approach.A tobacco NDPK gene was obtained and designated as NtNDPK1.NtNDPK1 is 704 bp in length and en- codes a putative 16.2 kD protein of 148 amino acids.Phylogenic analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type I (NDPK1).RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 ex- pression in roots,stems,leaves and buds.And expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl,but not significantly affected by Paraquat,wounding and low temperature (4℃) treatments,indicating that NtNDPK1 may play a certain role in response to abiotic stress.In vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphoryLation activity.     

A collision in 2009 as the origin of the debris trail of asteroid P/2010 A2

The peculiar object P/2010?A2 was discovered in January 2010 and given a cometary designation because of the presence of a trail of material, although there was no central condensation or coma. The appearance of this object, in an asteroidal orbit (small eccentricity and inclination) in the inner main asteroid belt attracted attention as a potential new member of the recently recognized class of main-belt comets. If confirmed, this new object would expand the range in heliocentric distance over which main-belt comets are found. Here we report observations of P/2010?A2 by the Rosetta spacecraft. We conclude that the trail arose from a single event, rather than a period of cometary activity, in agreement with independent results. The trail is made up of relatively large particles of millimetre to centimetre size that remain close to the parent asteroid. The shape of the trail can be explained by an initial impact ejecting large clumps of debris that disintegrated and dispersed almost immediately. We determine that this was an asteroid collision that occurred around 10 February 2009.     

Alternative splicing and expression of the insulin-like growth factor （IGF-1） gene in osteoblasts under mechanical stretch

Insulin-like growth factor 1 (IGF-1) promotes osteoblasts differentiation and bone formation, and its expression is induced by mechanical stretch, thus IGF-1 has been considered an effector molecule that links mechanical stimulation and local tissue responses. In this study, a mechanical stretching device was designed to apply physiological level static or cyclic stretching stimulation to osteoblasts. Different isoforms of IGF-1 mRNA were amplified by RT-PCR from the cells using respective primers and these amplified products were sequenced. An iso-form of IGF-1 splicing product was found to be selectively produced by osteoblasts under stretching stimulation. This IGF-1 isoform had identical sequence with the mechano growth factor (MGF) which was originally identified in muscle cells. Regulations of the expression of the liver-type IGF (L.IGF-1) and MGF in osteoblasts under stretch stimulation were further studied using semi-quantitative RT-PCR. Stretch stimulation was found to promot the expression of IGF-1 (L.IGF-1 and MGF), and for both isoforms expression was more effectively stimulated by cyclic stretch than static stretch. MGF was detected only in osteoblasts subjected to mechanical stretch, suggesting MGF was a stretch sensitive growth factor. Expression of MGF peaked earlier than that of L.IGF-1, which was similar to their regulation in muscle and suggested similar roles of MGF and L.IGF-1 in bone as in muscle cells. The functions of MGF and LIGF-1 in osteoblasts shall be established by further experimental studies.