A novel basic fibroblast growth factor delivery system fabricated with heparin-incorporated fibrin–fibronectin matrices for repairing rat sciatic nerve disruptions

Autologous nerve grafts are widely used in bridging critical gaps of peripheral nerves, but they remain associated with high morbidity of the donor site and lack of full recovery. As an alternative, we have focused on chitosan nerve conduits filled with a heparin-incorporated fibrin–fibronectin matrix serving as delivery systems for basic fibroblast growth factor (bFGF). The artificial nerve conduits were used for repairing sciatic nerve defects of 10 mm in adult rats. Three months post-operation, the conduction velocity recovery index (CVRI) and the muscle restoration rate (MRR) in animals of the experimental group were 32 ± 4.1 and 77.4 ± 7.9%, respectively, which were significantly higher than those of the PBS control group (17.8 ± 1.9 and 66.7 ± 6.5%), and similar to those of the autograft group (38.4 ± 3.9 and 81.3 ± 7.8%). These results were also consistent with the densities of regenerated axons in the three groups, which were demonstrated by histomorphological analysis.     

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.     

Mapping of qGL7-2,a grain length QTL on chromosome 7 of rice

A residual heterozygous line(RHL)carrying a heterozygous segment between two SSR loci RM11 and RM134 on the rice chromosome 7 was selected from a set of recombinant inbred lines from the cross D50(javanica)/HB277(indica).The former parent produces much longer grains than the latter.Selfed progenies of this selection were analyzed genotypically(SSRs)and phenotypically(grain length).Grain length was discontinuously variable in the mapping populations,allowing for the placement of this QTL qGL7-2 within a～4.8 cM interval defined by RM351 and RM234.A set of new markers within this region were developed,which narrowed the QTL to a 278 kb region defined by the markers Indel1 and RM21945.This region contains 49 predicted genes.The results also suggest that the novel allele for grain length will be used for the application of marker assisted selection for the improvement of grain length.     

Molecular characterization and bioactivity of a CXCL13 chemokine in large yellow croaker Pseudosciaena crocea

A CXCL13-like chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCXCL13). The full-length cDNA of LycCXCL13 is 796 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 10.7 kDa. The deduced LycCXCL13 contains a 24-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines (C²⁵, C²⁷, C⁵² and C⁶⁸). It shares 35, 36 and 39% aa sequence identities to green puffer CXCL13-like, Atlantic salmon CXCL13 and Japanese flounder CXCL13 chemokines, and 24–29% identities to CXCL13 chemokines in mammals, respectively. Phylogenetic analysis showed that LycCXCL13 is more closely related to the CXCL13 subgroup than to any other CXC chemokine subgroups. LycCXCL13 gene was constitutively expressed in all tissues examined, except for intestine. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL13 gene expression was significantly up-regulated in spleen, head kidney, heart and gills at 24 h post-injection. Real-time PCR results showed that LycCXCL13 gene expression reached peak level in spleen and head kidney at 12 h after induction by poly(I:C), while its expression increased to the highest level in head kidney at 24 h or in spleen at 48 h by bacterial vaccine. Recombinant LycCXCL13 protein produced in E. coli BL21 exhibited obvious chemotaxis to the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggest that LycCXCL13 may be involved in inflammatory responses as well as homeostatic processes in large yellow croaker.     

Physical properties and biocompatibility of a porous chitosan-based fiber-reinforced conduit for nerve regeneration

Porous fiber-reinforced chitosan nerve conduits were fabricated from chitosan yarns and a chitosan solution by combining an industrial braiding method with a mold casting/lyophilization technique. The conduits were permeable to molecules ranging in molecular size from 180 Da (glucose) to 66,200 Da (BSA). The compressive load of the reinforced conduits was significantly higher than that of a non-reinforced control conduit at equal levels of strain. The tensile strength of the reinforced conduits was also increased from 0.41 ± 0.17 to 3.69 ± 0.64 MPa. An in vitro cytotoxicity test showed the conduits were not cytotoxic to Neuro-2a cells. Preliminary in vivo implantation testing indicated that the conduits were compatible with the surrounding tissue. Aijun Wang and Qiang Ao contributed equally to this work.     

Mutations in HOXD13 underlie syndactyly type V and a novel brachydactyly-syndactyly syndrome

HOXD13, the homeobox-containing gene located at the most 5' end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores >3 (theta =0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A-->G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes--syndactyly type V and brachydactyly type A4--and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations.     

Comparative study of C-Reactive Protein and other biochemical parameters in patients with hepatitis B and malaria in Calabar, Nigeria

Serum levels of C-reactive proteins (CRP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), total protein, albumin and globulins were investigated using high sensitivity Immunoturbidometric and colorimetric techniques in individuals with hepatitis (n=50), Malaria (n=50) and 40 control subjects in age range of 30 to 65 years. The hepatitis patients had a significantly higher (P < 0.01) level of aminotransferases when compared to malaria patients and control subjects. The mean value of ALT was 103.50 ± 71.4 IU/L and 46.72 ±17.48 IU/L for hepatitis and malaria respectively. The values for AST were 116.76 ± 63.27 IU/L and 57.74 IU/L ± 15.18 IU/L for hepatitis and malaria respectively while the values for control were 34.75 ± 14.64 and 35.25 ± 15.56 IU/L for AST and ALT respectively. The malaria patients showed a significantly higher level (P < 0.01) of aminotransferases when compared to the control. The mean serum CRP levels were 0.71 ± 0.11 mg/dL and 0.78 ± 0.13 mg/dL for hepatitis and malaria respectively. These values were significantly higher (P < 0.01) than those of the controls which was 0.32 ± 0.12 mg/dL. The values of CRP in malaria were significantly higher (P< 0.05) when compared with hepatitis. In malaria, AST correlated with CRP (r = 0.58). The mean serum proteins of hepatitis patients were significantly lower (P < 0.05) than those of the control and malaria while there were no significant differences between the total protein in malaria when compared with control. Albumin levels in both patients were significantly lower (P > 0.05) than those of the controls. The mean values were 33.40 ± 3.40g/L and 34.47 ± 3.56g/L for hepatitis and malaria respectively and 37.00 ± 3.43 g/L for the control. C-reactive protein correlated negatively with albumin in malaria (r = -0.26) while albumin had a negative correlation with globulin(r = -0.36). Also albumin-globulin ratio were significantly (P < 0.05) decreased in both patients when compared with controls. This result suggests that a systemic acute phase response is present in hepatitis and malaria patients hence measurement of C-reactive proteins may be helpful in the diagnosis and management of hepatitis and malaria; especially in the malaria endemic region such as Nigeria. Keywords: Hepatitis B, Malaria, C-reactive protein, Liver function tests.     

大肠杆菌苹果酸合酶A的酶学和生理功能研究

苹果酸合酶是乙醛酸循环的关键酶之一。E.coli中苹果酸合酶A(malate synthase A,MSA)由aceB基因编码。根据E.coli基因组序列设计引物,利用PCR技术扩增aceB基因,并将其克隆入pET-29b(+),构建了重组表达质粒pET-MSA。经IPTG诱导,MSA在E.coliRosetta(DE3)中获得高效表达。纯化的MSA蛋白的分子量大小约为60 kDa,最适反应pH值和最适温度分别是pH值8.0、30℃。纯化的蛋白质在Mg2+存在时才能发挥最大的活性,其对乙酰辅酶A的Km和Vmax分别是8.07μM和3.6μM/min。此外构建了MSA和苹果酸合酶G(MSG)基因敲除菌株MG::ΔaceB和MG::ΔaceBΔglcB。研究发现缺少MSA的E.coli突变菌株在乙酸中的生长速率要比野生型菌株慢很多,表明MSA对大肠杆菌在乙酸中的生长起着重要作用。MSG虽然能部分补偿MSA的作用,但是包含MSA的乙醛酸旁路是更有效的乙醛酸代谢途径。     

Quantitative determination of buagafuran in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of buagafuran in human plasma. The analyte was extracted from plasma samples with hexane after addition of isotopic internal standard and chromatographed on a RP-C(8) column. The mobile phase consisted of methanol-water (90:10, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.5-200 ng/mL. Inter- and intra-day precision (RSD%) were all within 15% and the accuracy (RE%) was equal or lower than 9.5%. The lower limit of quantitation (LLOQ) was 0.5 ng/mL. The extraction recovery was on average 38.1% and the detection was not affected by the matrix. The method was successfully applied to the pharmacokinetic study of buagafuran in healthy Chinese volunteers.