[Risk factors for cow's milk protein allergy in infants: a multicenter prospective nested case-control study]. / å©´å„¿ç‰›å¥¶è›‹ç™½è¿‡æ•çš„å±é™©å› ç´ ï¼šä¸€é¡¹å¤šä¸­å¿ƒå‰çž»æ€§å·¢å¼ç— ä¾‹å¯¹ç §ç ”ç©¶.

OBJECTIVES: To explore the risk factors associated with cow's milk protein allergy (CMPA) in infants. METHODS: This study was a multicenter prospective nested case-control study conducted in seven medical centers in Beijing, China. Infants aged 0-12 months were included, with 200 cases of CMPA infants and 799 control infants without CMPA. Univariate and multivariate logistic regression analyses were used to investigate the risk factors for the occurrence of CMPA. RESULTS: Univariate logistic regression analysis showed that preterm birth, low birth weight, birth from the first pregnancy, firstborn, spring birth, summer birth, mixed/artificial feeding, and parental history of allergic diseases were associated with an increased risk of CMPA in infants (P<0.05). Multivariate logistic regression analysis revealed that firstborn (OR=1.89, 95%CI: 1.14-3.13), spring birth (OR=3.42, 95%CI: 1.70-6.58), summer birth (OR=2.29, 95%CI: 1.22-4.27), mixed/artificial feeding (OR=1.57, 95%CI: 1.10-2.26), parental history of allergies (OR=2.13, 95%CI: 1.51-3.02), and both parents having allergies (OR=3.15, 95%CI: 1.78-5.56) were risk factors for CMPA in infants (P<0.05). CONCLUSIONS: Firstborn, spring birth, summer birth, mixed/artificial feeding, and a family history of allergies are associated with an increased risk of CMPA in infants.

[Effect of superfine powder and aqueous extract of Polygonati Rhizoma on rats with natural perimenopausal syndrome].

This study aims to examine the effect of superfine powder and aqueous extract of Polygonati Rhizomaon on natural perimenopausal syndrome in rats and explore the underlying mechanism. To be specific, a total of 60 female SD rats(14-15 months old) with estrous cycle disorder were screened by the vaginal smear and randomized into model control group, ß-estradiol 3-benzoate group(0.1 mg·kg~(-1)), superfine powder of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)) and aqueous extract of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)), and another 10 female SD rats(14-15 months old) were selected as the youth control group. The administration lasted 6 weeks. Then the perimenopausal syndrome-related indexes such as body temperature, microcirculatory blood flow of face and ear, vertigo period, salivary secretion, grip force, and bone strength were determined and open field test was conducted. The immune system-related indexes such as the wet weight and index of thymus and spleen, percentage of T lymphocytes and subgroups in peripheral blood, and hematological indexes were measured. In addition, the ovary-related indexes such as estrous cycle, the wet weight and index of uterus and ovary, ovarian tissue morphology, and cell apoptosis were determined. Moreover, hypothalamus-pituitary-ovary axis(HPO)-related indexes such as serum sex hormone levels, cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and cytochrome P450 family 17 subfamily A member 1(P450 17A1) in ovarian tissue were measured. The results showed that the superfine powder and aqueous extract of Polygonati Rhizoma significantly decreased body temperature(anal, facial and dorsal temperature), microcirculatory blood flow in the ear, and vertigo period, increased salivary secretion, grip force, bone strength, total distance and total speed in the open field test, wet weight and index of thymus and spleen, lymphocyte ratio, CD3~+ level, and CD4~+/CD8~+ ratio, reduced neutrophil number and ratio, estrous cycle disorder ratio, and number of ovarian apoptotic cells, raised wet weight and index of uterus, wet weight of ovary, levels of inhibin B(INHB), estradiol(E_2), anti-müllerian hormone(AMH), and ovarian CYP11A1 and CYP19A1, decreased follicle-stimulating hormone(FSH) and luteinizing hormone(LH) content, and improved ovarian tissue morphology. It is suggested that the superfine powder and aqueous extract of Polygonati Rhizoma can improve the symptoms associated with natural perimenopausal syndrome in rats and enhance ovarian function and immune function. The mechanism is that they regulate HPO axis function by increasing estrogen synthesis.

[Optimization of expression and purification of recombinant Salvia miltiorrhiza WRKY1 protein in Escherichia coli].

WRKY transcription factor is one of the Zinc finger proteins which contains a highly conserved WRKY domain and is a family of the plant-specific transcription factor. The plasmid pET28a-SmWRKY1 harboring Salvia miltiorrhiza WRKY1 (SmWRKY1) gene was successfully transformed and expressed in Escherichia coli BL21 (DE3). The conditions on protein expression of SmWRKY1 in E. coli, including induction duration, temperature, IPTG concentration and the E. coli concentration were optimized. The results showed that the highest protein expression of SmWRKY1 was obtained at 24 hours after the E. coli was cultured in the presence of 0.2 mol x L(-1) IPTG at 20 degrees C with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g x L(-1) as inclusion body, which was first extracted using urea, and then purified by Ni2+ affinity chromatography and identified by SDS-PAGE. The expression of SmWRKY1 in E. coli was further confirmed by western blotting analysis.

[Construction of Saccharomyces cerevisiae whole-cell biocatalyst system for conversion miltiradiene].

Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases (SmCPR1) from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.

[Expression analysis and subcellular localization of an ERF transcription factor from Salvia miltiorrhiza].

OBJECTIVE: A SmERF1 gene was isolated from Salvia miltiorrhiza, and expression patterns to different stress condition were analysed in the root tissues of S. miltiorrhiza. METHOD: The cDNA of SmERF1 gene from S. miltiorrhiza was isolated by RTPCR, and the phylogenetic tree using the neighbour-joining tree method in Mega 5 was obtained. To confirm the protein is likely to localize in the nucleus, the SmERF1 coding region was fused to the N-terminus of the GFP gene under the control of the CaMV 35S promoter and transferred into onion epidermal cells using the particle bombardment method. Semi-quantitative RT-PCR analysis revealed different expression pattern of SmERF1 gene in response to exogenous ABA, MeJA and SA. RESULT: The phylogenetic tree analysis revealed that SmERF1 is most similar to AP2/ERF VII subgroup members. The transient expression of the SmERF1::GFP fusion protein indicated that the SmERF1 was exclusively localized to the nucleus. The transcript of SmERF1 highly accumulated when the plants were treated with MeJA, while accumulated slightly in response to exogenous ABA, salicylic acid. CONCLUSION: These results suggest hormone such as ABA, MeJA and SA signaling pathways can be involved in the activation and inhibition of the SmERF1.

[Optimizing expression of recombinant jasmonate ZIM-domain protein from Salvia miltiorrhiza].

OBJECTIVE: Accumulation of tanshinton in Salvia miltiorrhiza are enhanced by exogenous application of jasmonates. The core JA signaling module COI1/JAZ/MYC2 play a central role on control of downstream gene expression in the JA pathway. To obtained the antibody of SmJAZ, SmJAZ recombinant protein was expressed in Escherichia coli and optimal expression was performed. METHOD: The full-length SmJAZ1 ORF was sub-cloned in a prokaryotic expression vector pET32a. The recombinant fusion protein had high expression level in BL21 (DE3) strain of E. coli, and SDS-PAGE analysis showed its molecular weight was about 24 kDa. RESULT: The induction of E. coli [pET32-JAZ1] in different temperature, induction time, IPTG concentrations and IPTG adding time of E. coli were performed. The induction time and the induction temperature are positively related trends with SmJAZ1 protein expression, and IPTG concentration had no significant impact in protein expression, whereas IPTG adding time had significant impact on protein expression. CONCLUSION: Shaking the culture at 30 degrees C until the A600 is approximately 0.9 (2 h in LB), and add IPTG to a final concentration of 0.1 mmol x L(-1), and then the optimal expression of SmJAZ1 recombinant protein were accumulated after the induction time of 20 h.

A developmental switch between electrical and neuropeptide communication in the ventromedial hypothalamus.

Dissecting neural connectivity patterns within local brain regions is an essential step to understanding the function of the brain.1 Neural microcircuits in brain regions, such as the neocortex and the hippocampus, have been extensively studied.2 By contrast, the microcircuit in the hypothalamus remains largely uncharacterized. The hypothalamus is crucial for animals' survival and reproduction.3 Knowledge of how different hypothalamic nuclei coordinate with each other and outside brain regions for hypothalamus-related functions has been significantly advanced.4-9 Although there are limited studies on the neural microcircuit in the lateral hypothalamus (LHA)10,11 and the suprachiasmatic nucleus (SCN),12,13 the patterns of neural microcircuits in most of the given hypothalamic nuclei remain largely unknown. This study applied combinatory approaches to address the local neural circuit pattern in the ventromedial hypothalamus (VMH) and other hypothalamic nuclei. We discovered a unique neural circuit design in the VMH. Neurons in the VMH were electrically coupled at the early postnatal stage like ones in the neocortex.14 However, unlike neocortical neurons,14,15 they developed very few chemical synapses after the disappearance of electrical synapses. Instead, VMH neurons communicated with neuropeptides. The similar scarceness of synaptic connectivity found in other hypothalamic nuclei further indicated that the lack of synaptic connections is a unique feature for local neural circuits in most adult hypothalamic nuclei. Thus, our findings provide a solid synaptic basis at the cellular level to understand hypothalamic functions better.

Effects of Dendrobium officinale ultrafine powder on sub-health mice induced by unhealthy lifestyle based on neuroendocrine immune system.

Sub-health status, in which a person's mind and body exist in a low-quality state of being between disease and health, has become an urgent public health problem that cannot be ignored globally. One of the most apparent sub-health symptoms is fatigue, and it also shows a significant decrease in mental vitality and adaptability caused by disruption of the neuroendocrine-immune system. Dendrobium officinale (DOF) has a long history of use in China as a medicinal food with immune-regulating, anti-fatigue, anti-oxidant, and hypoglycemic effects. The ameliorative effects of Dendrobium officinale on sub-health mice are investigated in this present study, as well as its underlying mechanisms via neuroendocrine-immune (NEI) modulation. Forty male KM mice were divided into normal control group (NC), model control group (MC), and two doses of ultrafine DOF powder (DOFP) intervention groups: DOFP-L (0.1 g kg-1), DOFP-H (0.2 g kg-1) groups. Sub-health mice were induced by mimicking unhealthy human lifestyles, including cold water swimming, limbs restriction, an unhealthy diet, and sleep deprivation for seven weeks. The findings revealed that DOFP intervened sub-health mice have less bodyweight loss, normal fecal morphology, as well as lower face temperature and blood flow, which is similar to the normal mice. Moreover, sub-health mice treated with DOFP showed improved forelimb grip strength and exercise endurance in weight-loaded exhaustion swimming and cold water exhaustion swimming, combined with reduced content of lactic acid (LD), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) in the plasma, increased storage of liver glycogen (LG), and muscle glycogen (MG), as well as increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), reduced malondialdehyde (MDA) levels in the liver. Additionally, DOFP could increase the counts of autonomous movements of sub-health mice, minimize tail suspension time, and perform well in the elevated plus maze and open field tests, all of which are associated with anti-depression and anti-anxiety. Moreover, mechanistic investigations revealed that DOFP could alleviate plasma corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and cortisol (CORT) related hormones in the HPA axis, increase the level of hypothalamic norepinephrine (NE) and plasma ß-endorphin (ß-EP) of sub-health mice, while downregulating the content of 5-hydroxytryptamine (5-HT), dopamine (DA), and the relative mRNA expression of 5-HT1A and CRH in hypothalamus, and increase immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), and CD4+/CD8+ T cell ratio levels. In conclusion, DOFP can relieve symptoms such as fatigue and depression in sub-health mice by regulating the disorder of the neuroendocrine-immune network.

Buddleoside-Rich Chrysanthemum indicum L. Extract has a Beneficial Effect on Metabolic Hypertensive Rats by Inhibiting the Enteric-Origin LPS/TLR4 Pathway.

As the number of patients with metabolic hypertension (MH) is increasing, there is an essential require for global measures to prevent and treat MH. Flavonoids such as buddleoside (BUD) from Chrysanthemum indicum L. are the main pharmacological components of cardiovascular activities. Previous studies have suggested that the buddleoside-rich Chrysanthemum indicum L. extract (BUDE) can reduce blood pressure in spontaneously hypertensive rats (SHR). However, its effect on MH and how it works remains to be researched. In this study, it was observed that BUDE could lower blood pressure, improve dyslipidemia, and decrease the level of plasma LPS in MH rats. Moreover, BUDE improved intestinal flora and increased the expression of occludin and claudin-1 in the colon, and improved the pathological injury of the colon. Western bolt and qRT-PCR experiments showed that BUDE could down-regulate TLR4 and MyD88 protein and mRNA expression and inhibit phosphorylation of IKKß, IκBα and NF-κB p65 in vessels of MH rats. These results showed that BUDE could regulate intestinal flora, improve intestinal barrier function, reduce the production and penetration of LPS, thereby inhibiting the vascular TLR4/MyD88 pathway, improving vascular endothelial function, and ultimately lowering blood pressure in MH rats. This study provides a new mechanism of BUDE against MH by inhibiting the enteric-origin LPS/TLR4 pathway.

[Insulin-like growth factor 1 levels and their association with growth and development in infants aged 1-24 months].

OBJECTIVE: To study serum insulin-like growth factor 1 (IGF-1) levels and their association with growth and development in infants aged 1-24 mouths. METHODS: A total of 525 healthy infants (125 preterm, 400 term) were enrolled. Serum IGF-1 levels were measured using ELISA 1.5, 4, 6, 8, 12, 18 and 24 months after birth. The body weight and body length were simultaneously measured. RESULTS: Serum IGF-1 levels were the lowest in preterm infants 1.5 months after birth (86+/-60 ng/mL). Thereafter, serum IGF-1 levels increased, and were significantly higher than those in term infants between 4 and 12 months after birth. Serum IGF-1 levels in term infants were the highest (116+/-52 ng/mL) 1.5 months after birth during their life of 12 months old. Thereafter, serum IGF-1 levels decreased and reached to a nadir (69+/-58 ng/mL) 8 months after birth. IGF-I levels were positively correlated with the weight and the height (SDS) in both preterm and term infants. CONCLUSIONS: Serum IGF-1 levels are closely associated with growth and development in infants.

Diagnostic roles of urinary kidney microvesicles in diabetic nephropathy.

BACKGROUND: The pathology of diabetic nephropathy (DN) broadly involves the injury of glomeruli, tubulointerstitium and endothelium. Cells from these compartments can release increased numbers of microvesicles (MVs) into urine when stressed or damaged. Currently whether urinary MVs from these three parts can help diagnose DN and reflect pathological features remain unclear. METHODS: Forty-nine patients with histologically proven DN and 29 proteinuric controls with membranous nephropathy or minimal change disease were enrolled. Urinary podocyte, proximal tubular and endothelial cell-derived MVs were quantified by flow cytometry. Renal glomerular, tubulointerstitial and vascular lesions were semi-quantitatively scored and their relevance to urinary MVs were analyzed. RESULTS: DN patients had greater numbers of urinary MVs from podocytes, proximal tubular and endothelial cells compared with proteinuric controls. The combination of podocyte nephrin+ MVs and diabetic retinopathy optimally diagnose DN with 89.7% specificity and 88.9% sensitivity. Moreover, positive correlations were observed between urinary levels of proximal tubular MVs and the severity of tubular injury and between urinary levels of endothelial MVs and the degree of vascular injury. Using urinary proximal tubular MVs as the indicators for tubular injury, the differences between DN patients and proteinuric controls diminished after matching the degree of renal vascular injury or when proteinuria >8 g/24 h. CONCLUSIONS: Urinary kidney-specific cell-derived MVs might serve as noninvasive biomarkers for the diagnosis of DN in diabetic proteinuric patients. Their elevated levels could reflect corresponding renal pathological lesions, helping physicians look into the heterogeneity of DN.

Interstitial eosinophilic infiltration in diabetic nephropathy is indicative of poor prognosis, with no therapy benefit from steroid.

BACKGROUND: Studies suggested that eosinophils in diabetes might be associated with severity of diabetic nephropathy (DN). In a retrospective study of 102 Chinese patients with biopsy-proven DN, we aimed to evaluate relationships of both blood and renal eosinophils (Eos) to the severity of DN and check whether Eos can serve as an indicator of prognosis as well as the therapeutic effect of steroids. METHODS: One hundred and two patients diagnosed with DN were enrolled. Demographical and clinical data and histopathological scores were associated. Interstitial eosinophilic aggregates (IEA) were defined as the presence of ≥10 Eos in at least one high-power field. End-stage renal disease was defined as the end point. RESULTS: We observed that log2 (blood eosinophil counts) correlated with neutrophil counts, proteinuria, and tubulointerstitial inflammatory cell infiltration. IEA was observed in 33.3% of the DN patients and was associated with decreased estimated glomerular filtration rate, higher proteinuria, hematuria, higher HbA1c, increased blood eosinophil counts, tubular injury, tubulointerstitial chronicity, and interstitial inflammation. IEA was associated with worse renal prognosis (hazard ratio [HR] 2.424, P = 0.008). Consistently, urine eosinophil cationic protein (ECP) (ng/mgCr) was associated with renal injury and poor renal prognosis (HR 1.173, P = 0.020). Patients with IEA were more likely to be treated with steroid/immunosuppressants (47.1% vs 14.7%, P = 0.001) but did not show renal benefit. CONCLUSIONS: It suggested that both blood and renal infiltrated eosinophils were prevalent in DN and associated with severity of DN. IEA in renal pathology showed better fit in correlation with renal prognosis. Treatment with steroid/immunosuppressants showed no significant improvement regarding renal prognosis.

Potential adjuvantic properties of innate immune stimuli.

Toll-like receptors (TLRs) are a family of conserved pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns and serve as primary sensors of the innate immune system. Ten members of the TLR family have so far been identified in the human genome. The ligands for these receptors are structurally highly conserved microbial molecules such as lipopolysaccharides (LPS) (recognized by TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double-stranded RNA (TLR3), CpG motif-containing DNA (TLR9) and profilin present on uropathogenic bacteria (TLR 11). Complementing the TLRs are the nucleotide-binding domain (NOD), leucine rich repeat containing family (or Nod-like Receptors, NLRs), which detect muramylpeptides released from bacterial peptidoglycan (PGN) in the intracytoplasmic compartment, as well as the retinoic-acid-inducible protein 1 (RIG-I-like receptors; RLRs) which sense single-stranded RNA of viral origin. The activation of PRRs by their cognate ligands leads to production of inflammatory cytokines, upregulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer cells, in addition to priming and amplifying antigen-specific T-, and B-cell effector functions. Thus, these stimuli serve to link innate and adaptive immunity and can therefore be exploited as powerful adjuvants in eliciting both primary and anamnestic immune responses. This review summarizes what is currently known about the immunopotentiatory and adjuvantic activities of innate immune stimuli.

[Inhibitory effects of pretreatment with Buyanghuanwu decoction on inflammatory cytokine expressions in the kidneys of rats after induction of brain death].

OBJECTIVE: To observe the inhibitiory effects of pretreatment with Buyanghuanwu decoction (BYHWT) on inflammatory cytokine expressions in the kidneys and the level of reactive oxygen species (ROS) by peripheral blood neutrophils of rats after induction of brain death (BD), and to investigate the effect of BYHWT on the improvement of kidney quality from BD donor. METHODS: 30 male Wistar rats were randomly divided into 3 groups: control group, BD model group and BYHWT group. 6 hours after successful onset of brain death,only the BD rats whose mean arterial blood pressure were higher than 80 mmHg were accepted as donors. Kidneys were harvested and peripheral blood was taken from BD rats. RT-PCR was used to detect the expressions of TNF-a and IL-lpfl mRNA. Western blot was adopted to analyze the expressions of both TNF-alpha and IL-lp protein,and the expression of phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK). Reactive oxygen species( ROS) in the peripheral blood neutrophils were labeled with CM-H2DCFDA and then detected with Flow Cytometry. RESULTS: The expressions of both TNF-alpha and IL-1beta mRNA and protein, and the p-p38MAPK proteins all significantly increased in BD group compared with control group (P < 0.01). However, those in BYHWT group statistically decreased compared with BD group (P < 0.05), but they significantly increased in comparison with control group (P < 0.01). There was a close relation between the expression of p-p38MAPK protein and the expressions of both TNF-a and IL-1beta mRNA and protein. ROS level significantly increased in BD group (P < 0.05 ), whereas it significantly decreased in BYHWT group (P < 0.05). There was no statistically significant difference between BYHWT group and control group (P > 0.05). CONCLUSION: Pretreatment of the rats with BYHWT prior to the induction of rat brain death, can significantly suppress the expressions of inflammatory cytokines and ROS level in the kidneys of rats from BD. It might be related to the blockage of key target points in p38MAPK signaling pathway. Therefore pretreatment with BYHWT could hopefully be an ideal way to improve the quality of kidneys from brain dead donors prior to transplantation.

Human papillomavirus genotypes and p16 expression in oral leukoplakia and squamous cell carcinoma.

Several studies have shown a broad variation in the prevalence of human papillomavirus (HPV) in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), whereas the relationship is less well-defined and specific HPV genotypes lack examination in OLK. In the present study, the role of HPV and surrogate p16 expression was investigated to explore the correlation and pathogenesis in OLK and OSCC. Polymerase chain reaction (PCR) and flow-through hybridization technology were utilized to detect HPV genotypes in oral exfoliated cells from 30 healthy volunteers, 103 OLK and 30 OSCC patients. Expression of p16 was assessed by immunohistochemistry (IHC) in biopsies from these OLK and OSCC, in addition to 15 normal oral mucosal tissues as the control group. The healthy controls showed 3.3% (1/30) HPV presence; In OLK and OSCC, the detection rate was 4.9% (5/103), 3.3% (1/30), respectively. No significant relationship between HPV and OLK or OSCC was observed when compared with the control group (P>0.05). All 6 HPV-positive OLK and OSCC cases had p16 overexpression. But the sensitivity of p16 IHC was poor, because 88.4% (38/43) of p16 over-expressed OLK were HPV negative. There was no statistical significance between HPV and the sex, age, site, alcohol consumption, or smoking. These findings suggested HPV had a low prevalence in OLK and OSCC. This suggests the detection of HPV genotypes by PCR in exfoliated cells combined with p16 IHC may be more accurate to represent HPV infection.

Molecular cloning, expression and characterization of three short chain alpha-neurotoxins from the venom of sea snake--Hydrophiinae Hydrophis cyanocinctus Daudin.

Three different genes named sn311, sn316 and sn285 were discovered by large-scale randomly sequencing the high quality cDNA library of the venom glands from Hydrophiinae Hydrophis cyanocinctus Daudin. Sequence analysis showed that these three genes encoded three different short chain alpha-neurotoxins of 81 amino acids, which contained a signal peptide of 21 amino acids and followed by a mature peptide of 60 amino acids. Amino acid comparison reveals that mature peptides of sn311 and sn316 are highly homologous, with the only variance at position 46, which is Lys46 and Ser46, respectively. Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain alpha-neurotoxins, Asp31 and Arg33. The coding sequences of three neurotoxins were cloned into a thioredoxin (TRX) fusion expression vector (pTRX) and expressed as soluble recombinant fusion proteins in E. coli. After purification, approximately 10 mg/l recombinant proteins with the purity up to 95% were obtained. These three recombinant proteins are designated as rSN311, rSN316 and rSN285, they have a molecular weight of 6.963, 6.920 and 6.756 kDa, respectively, which are similar to those predicted from amino acid sequences. LD50 values of rSN311, rSN316 and rSN285 are 0.0827, 0.095, and 0.0647 mg/kg to mice, respectively. Studies on effects of these recombinant proteins on neuromuscular transmission were carried out, and results indicate that they all can produce prompt blockade of neuromuscular transmission, but display distinct biological activity characteristic individually. The results from UV-circular dichroism (CD) spectra indicate that they share similar secondary structure compared to other identified alpha-neurotoxins, and no significant structural differences in these recombinant proteins are observed.

Functional expression and characterization of a recombinant phospholipase A2 from sea snake Lapemis hardwickii as a soluble protein in E. coli.

Three full-length phospholipase A(2) (PLA(2)) cDNAs from sea snake Lapemis hardwickii venom were cloned and sequenced in our previous study. In order to investigate their biological functions, we established a fusion expression system for PLA(2)-9 in E. coli. The open reading frame encoding mature peptide of PLA(2)-9 was subcloned into the vector pTRX. The Trx-PLA(2)-9 fusion protein was expressed as a soluble protein by IPTG induction at 23 degrees C. The fusion protein was purified with metal-chelate affinity chromatography and then cleaved by enterokinase. The mature recombinant PLA(2)-9 was further purified by ion-exchange chromatography and a final yield of approximately 2.5mg pure PLA(2)-9 from 1l of bacteria culture was obtained. The catalytic activity of recombinant PLA(2)-9 (rPLA(2)-9) was measured and found to be similar to native enzyme. As the Austrelaps superbus PLA(2), which shares 90% nucleotide sequence similarity to PLA(2)-9, the rPLA(2)-9 displayed the anti-platelet aggregation effect. Site-directed mutagenesis of the two conserved residues, His-48 and Asp-49, resulted in the loss of catalytic activity, however did not affect the inhibition effect of platelet aggregation suggesting that these two activities of sea snake PLA(2)-9 may be dissociated.

Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3',5'-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C. The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (), having 61.7% beta-sheet and no alpha-helix. Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD(50) of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5+/-160.5%. The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.

Cloning and characterization of an acidic cytolysin cDNA from sea anemone Sagartia rosea.

A full-length cDNA of cytolysin (Src I) was isolated from the tentacle of Sagartia rosea (a representative species in China) by reverse transcription polymerase chain reaction. The cDNA with an open reading frame of 648 bp encodes a precursor protein of 216 amino acids, which contains a prepropeptide of 38 amino acids including a signal peptide of 19 amino acids and a propart of 19 amino acids. Lys-Pro at C-terminus of propart is a cleavage site for proline-endopeptidase-like protease. The mature cytolysin has a molecular mass of 19.6 kDa and a pI value of 4.8. Src I is an acidic cytolysin found in sea anemone and shares 75% amino acid sequence similarity to equinatoxin II (Eqt II). The predicted secondary structure of the mature cytolysin comprises 15% alpha-helix, 45% beta-sheet, and 40% random coil. The characteristic amphiphilic alpha-helix of cytolysin is located at the N-terminus of the processed Src I.

Identification and Funtional Characterization of Three Postsynaptic Short-chain Neurotoxins from Hydrophiinae, Lapemis hardwickii Gray.

Three cDNA clones, sn12, sn36 and sn160, encoding isoforms of postsynaptic short-chain neurotoxins, were cloned by screening a cDNA library of the venom from Hydrophiinae, Lapemis hardwickii Gray. The sequences of three cDNA clones encoded proteins consisting of 60 amino acid residues. There was only one amino acid substitution among the three isoforms SN12, SN36 and SN160 at the position 46 of mature proteins, and they were Pro(46), His(46) and Arg(46), respectively. The three molecules were expressed in Escherichia coli and the recombinant proteins were characterized. Different LD(50) were obtained, namely 0.0956 mg/kg, 0.3467 mg/kg and 0.2192 mg/kg, when the SN12, SN36 and SN160 were injected into Kunming mice(i.p.). In analgesic effect assayed by the acetic acid-induced writhing method, SN12 and SN160 showed similar analgesic effect, but SN36 had effects significantly different with the other two. Our studies suggested that the amino acid residues on position 46 could affect the combination between the postsynaptic short-chain neurotoxins and the nicotinic acetylchoine receptor, since different amino acid substitution resulted in different biological activities.