Basal Forebrain Cholinergic Deficits Reduce Glucose Metabolism and Function of Cholinergic and GABAergic Systems in the Cingulate Cortex

PURPOSE: Reduced brain glucose metabolism and basal forebrain cholinergic neuron degeneration are common features of Alzheimer's disease and have been correlated with memory function. Although regions representing glucose hypometabolism in patients with Alzheimer's disease are targets of cholinergic basal forebrain neurons, the interaction between cholinergic denervation and glucose hypometabolism is still unclear. The aim of the present study was to evaluate glucose metabolism changes caused by cholinergic deficits. MATERIALS AND METHODS: We lesioned basal forebrain cholinergic neurons in rats using 192 immunoglobulin G-saporin. After 3 weeks, lesioned animals underwent water maze testing or were analyzed by 18F-2-fluoro-2-deoxyglucose positron emission tomography. RESULTS: During water maze probe testing, performance of the lesioned group decreased with respect to time spent in the target quadrant and platform zone. Cingulate cortex glucose metabolism in the lesioned group decreased, compared with the normal group. Additionally, acetylcholinesterase activity and glutamate decarboxylase 65/67 expression declined in the cingulate cortex. CONCLUSION: Our results reveal that spatial memory impairment in animals with selective basal forebrain cholinergic neuron damage is associated with a functional decline in the GABAergic and cholinergic system associated with cingulate cortex glucose hypometabolism.

Cloning and expression of pokeweed antiviral protein gene from Phytolacca americana in Pichia pastoris and the study of apoptosis of human neuroglioma cells U251 induced by recombinant PAP / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To clone the pokeweed anti-viral protein (PAP) gene, to express it in Pichia pastoris, and to study the inhibitory effect of PAP on U251 in vitro.</p><p><b>METHODS</b>The cDNA sequence encoding PAP was cloned by Real-time PCR from Phytolacca americana. The recombinant PAP was subcloned into the expression vector pPICZaA and expressed in Pichia pastoris GSI15 after methanol induction. SDS-PAGE analysis showed that the expressed PAP existed in the yeast culture supernatant. The drug cytotoxicity to U251 cells was assessed using MTT assay and the obvious apoptotic nuclei of the tumor cells detected using the method of single cell gel electrophoresis.</p><p><b>RESULTS</b>The full-length PAP gene was cloned. The recombinant expression plasmid pPICZaA-PAP was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass (M) of the recombinant protein was about 35 kDa. The degradation of the genome of the apoptotic cells induced by PAP was detected using the method of single cell gel electrophoresis. PAP possessed very high ability to inhibit the growth of U251. The anti-tumor activities (IC50) to U251 cells of PAP was 81.0 microg/mL.</p><p><b>CONCLUSION</b>PAP could be a potent anti-tumor candidate for inhibiting the growth of U251 and inducing its apoptosis.</p>

The effects of different PAP domains on hepatitis B virus replication / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of different PAP domains on hepatitis B virus replication.</p><p><b>METHODS</b>The full length and two truncated PAP mutants were cloned into a eukaryotic expression plasmid, and were transfected into HepG2.2.15 cells using lipofectamine 2000. 3 days after transfection, the medium and cells were collected. HBsAg and HBeAg were measured using ELISA. The titers of HBV DNA were quantified using fluorogenic quantitative PCR (FQ-PCR). HepG2 cells were used to determine the cytotoxicity of the plasmids transfection by MTT assays.</p><p><b>RESULTS</b>The inhibitory effect on HBV replication of the C-terminal 25 amino acids deleted PAP mutant (pXF3H-PAP14) was not significantly different from that of the full length PAP (pXF3H-PAP12) (Chi-square test = 0.5, 2.0, 0.02, probability value more than 0.05), however, the cytotoxicity of pXF3H-PAP14 was lower than that of pXF3H-PAP12 (Chi-square test = 7.7, probability value less than 0.01). Both N-terminal 69 amino acids deleted mutant and C-terminal 25 amino acids deleted mutant had no cytotoxicity and no antiviral activity.</p><p><b>CONCLUSION</b>C-terminal 25 amino acid of PAP is related to cytotoxicity but not related to antiviral activity of PAP. N-terminal 69 amino acid of PAP is related to the anti-HBV effect of PAP.</p>

Study on expression of curcin gene cloned from Jatropha curcas in Escherichia coli / 中国中药杂志

<p><b>OBJECTIVE</b>To construct mature protein curcin gene prokaryotic expression vectors in Escherichia coli and choose the optimal inducing condition of the recombinant strains.</p><p><b>METHOD</b>The gene encoding of curcin was amplified from the genome of Jatropha curcas seeds by PCR and cloned into the expression vectors pQE-30 and pET-32 obtaining recombinant vectors pQE-R and pET-R respectively. The two vectors were transferred into E. coli BL21 (DE3) and the recombinant strains PRM and PRB were attained respectively. PRM and PRB were induced by different revulsants and under different temperature and time.</p><p><b>RESULT</b>The gene encoding of mature protein curcin was amplified by PCR and the recombinant strains PRM and PRB were obtained.</p><p><b>CONCLUSION</b>The results showed that PRB could not produce recombinant protein under such conditions. However, PRM could highly produce recombinant protein induced by 0.5 mmol x L(-1) IPTG at 28 degrees C for 6 h.</p>

Analysis on bioactivity of HIV-1 integrase by ELISA method / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop an ELISA-based method for analyzing biologic activities of HIV-1 integrase and for high throughput screening of integrase inhibitors.</p><p><b>METHODS</b>After expression, renaturation and purification of integrase, the bioactivity of integrase and the inhibition of luffin-a were evaluated with an in vitro assay based on biotin-avidin EILSA and chemiluminescent substrates.</p><p><b>RESULT</b>(1) The specific activity of the purified integrase was 54.92 units/mg of protein. (2)IC(50) (concentration causing 50% inhibition of integrase) of luffin-a was (0.63 +/- 0.026) micromol/L.</p><p><b>CONCLUSION</b>The non-radioactive assay can be used for analysis of bioactivities and high throughput screening of inhibitors of HIV-1 integrase.</p>

Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana / 生物工程学报

The cDNA sequence encoding pokeweed antiviral protein-II was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-II was subcloned into the expression vector pET-28a(+) and expressed in E. coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-II existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HIV-1 integrase to some extent (IC50 = 303 microg/mL, 220 microg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 s of 93 microg/mL and 102 microg/mL, respectively. The results suggested that pokeweed antiviral protein-II is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-II.

Cloning and expression of luffin-a gene from the seeds of Luffa cylindrical / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To clone luffin-a cDNA from the seeds of Luffa cylindrical, and to obtain bioactive recombinant luffin-a protein using the expression vector pET-44a (+) in E. coli.</p><p><b>METHODS</b>The cDNA sequence encoding luffin-a was cloned from the fresh seeds of Luffa cylindrical by RT-PCR. The target DNA fragments were sequenced after T-A cloning. The luffin-a expression plasmid was constructed by inserting the luffin-a cDNA fragment into vector pET-44a (+). Luffin-a was expressed in E. coli by addition of IPTG into final concentration 1.0 mmol/L. The recombinant luffin-a was identified by SDS-PAGE. The biological activity of luffin-a protein was evaluated by using the MTT assay in HepG2 cells following fluid-phase endocytosis.</p><p><b>RESULTS</b>In comparison with the reported luffin-a, the homology of nucleotide sequence of the cloned luffin-a gene was 99.73%, while their amino acid sequences were identical. The solubility of recombinant protein was analyzed by SDS-PAGE, and the luffin-a was mainly produced in inclusion bodies. The recombinant luffin-a, renatured by dialysis of the denatured products, showed a similar cytotoxicity to ricin A chain.</p><p><b>CONCLUSION</b>The cDNA of luffin-a has been successfully cloned. The recombinant luffin-a protein expressed by E. coli is bioactive.</p>

Role of positive charge of lysine residue on ribosome-inactivating property of gelonin.

The report that gelonin cross-linked with monoclonal antibodies with the use of 2-iminothiolane (2-IT) exhibited higher cytotoxicity than the conjugates prepared with the use of N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) alone, has prompted us to investigate the effect of epsilon-NH2 group modification with 2-IT on the ribosome-inactivating property (RIP) of gelonin. The purified gelonin was modified with 2-IT at a different molar ratio and their effects on immunoreactivity and ribosome-inactivating property were compared with those of N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (long chain-SPDP) and SPDP modified gelonin derivatives. Modification of single amino group with 2-IT results in about 25-50% inhibition of immunoreactivity and 60-70% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampers both immunoreactivity and protein synthesis inhibition property of gelonin. Both the long chain-SPDP with SPDP modifications showed more pronounced effects on immunoreactivity and RIP activity as compared to the similar ratio of 2-IT modification(s). It may, therefore, be concluded that the positive charge plays an important role in the immunological as well as the protein synthesis inhibitory effect of gelonin.

Purification and characterisation of gelonin from seeds of Gelonium multiflorum.

Gelonin, a ribosome-inactivating protein has been isolated from the seeds of Gelonium multifluorum of Euphorbiaceae family by two methods and the results are compared. In method-I conventional aqueous extraction, cation-exchange and gel-filtration chromatography has been used. In method-II S-Sepharose fast flow gel has been used to purify the proteins from the seed extract, followed by ammonium sulfate fractionation, cation-exchange and gel-filtration chromatography. Extensive physico-chemical and immunological characterizations show that molecular weight of gelonin as determined by gel-filtration chromatography and SDS-PAGE is approximately 30 kDa. The non-proteinous material which binds to CMC-gel in association with gelonin in method-I is substantially removed when gelonin is purified by method-II. Cation exchange, G-100 chromatography, RP-HPLC and SDS-PAGE show that method-II yields 50% more purified gelonin when compared to the yield by method-I. The immunoreactivity of gelonin obtained by methods I and II vary from 22-26% and 50-66% respectively and the ribosome-inactivating property vary from 46-56% and 70-87% respectively.

Spectroscopic characterization of gelonin--assignments secondary structure and thermal denaturation.

Gelonin, a type 1 ribosome inactivating protein (RIP), having only one polypeptide chain, and which could be used against deadly diseases like cancer and AIDS is investigated spectroscopically through infrared (IR), diffused reflectance infrared fourier transform (DRIFT) and Raman techniques and observed vibrational modes are assigned. It is found that gelonin is having mainly alpha-helix and beta-sheet structure with some turn and disordered structure, the estimated percentage structure using Raman data being approximately 32% alpha-helix, approximately 20% beta-sheet, approximately 26% turn and approximately 22% disorder type. The temperature dependent infrared study of gelonin reveals its thermal stability, the denaturation temperature being around 60 degrees C and disordered contribution increasing with heating.

Antiproliferative effect of basic fibroblast growth factor-saporin mitotoxin on keratocytes in culture

We evaluated the effect of the conjugate of basic fibroblast growth factor (FGF2) and saporin (FGF2-SAP) on proliferation of cultured keratocytes. Cultured rabbit and human keratocytes were incubated in medium containing 0.01 to 100 nM of chemical conjugate of EGF2 conjugated by disulfide bond to saporin (CCFS1), FGF2 genetically fused to saporin (rFGF2-SAP), FGF2, or saporin for three hours or four days and cell proliferation was quantified four days after the drug treatment. Proliferation of rabbit and human keratocytes was effectively inhibited by three hour and by four day exposure to CCFS1 and rFGF2-SAP in a dose-dependent manner, whereas it was affected minimally by four day exposure to saporin. Their inhibitory effects were detected at concentrations above 0.1 or 1 nM, and were most prominent in serum-stimulated rabbit keratocytes. These results suggest a potential role for FGF2-SAP in limiting proliferation of keratocytes during corneal wound healing.

Properties of a ribosome-inactivating protein, gelonin, purified using three different methods.

Ribosome-inactivating protein, gelonin, isolated from an Indian plant Gelonium multiflorum of Euphorbiaceae family has been used to design and synthesize immunotoxins and hormonotoxins for selective targeting purposes. Since gelonin isolated by aqueous extraction, cation-exchange chromatography and gel-filtration chromatography (Method I), contains non-proteinous material absorbing at 280 nm, the ammonium sulphate precipitation method (Method II) and Cibacron blue affinity chromatography method. (Method III) have been used to purify gelonin from the dry seeds. Three batches of gelonin purified by each method were prepared and subjected to extensive physico-chemical and immunochemical characterization. The molecular weight was determined by gel-filtration chromatography on a pre-calibrated Sephadex G-100, TSK-G4000 TW on HPLC or Superose-12 on fast protein liquid chromatography. In all cases, the molecular weight was approximately 30,000Da. The SDS-PAGE also revealed a homogeneous protein of 30kDa molecular weight. In Method II, the non-proteinous material which binds to CMC-gel in association of gelonin was substantially removed during ammonium sulphate fractionation. A careful analysis clearly revealed that Method II, although yielded low protein, gave gelonin devoid of the non-proteinous material. The SPDP modification of epsilon-NH2 groups of gelonin obtained from Methods I, II, and III was also carried out and its effect on immunoreactivity was studied.

Thermo-stability and antitumor activity on colon cancer cell lines of monoclonal anti-CEA antibody-saporin immunotoxin

Eight saporin peaks were obtained from the purification of seed extracts of Saponaria officinalis L. Saporin peak No. 6 (SAP-6) showed the highest activity in the inhibition of protein synthesis (98%) in an in vitro translation study. An immunotoxin (IT) was prepared from SAP-6 conjugated to a monoclonal anti-CEA antibody 26/5/1 (mab B) using N-succinimidyl pyridyl dithiopropionate (SPDP) and 2-iminothiolane as a cross linker. Under thermal stability study by a DSC (differential scanning calorimetry), the IT showed a denature temperature of 75 degrees C. In in vitro translation studies, the purified IT showed the same activity as SAP-6 at 10(-7) M and 10(-9) M protein concentration at 0, 30 and 60-min incubation effects with mab B and SAP-6 not conjugated at 24-hr incubation periods on human promyelocytic cell line HL 60 and on human colon adenocarcinoma cell lines which were SW 403, LoVo and LS 174 T. SAP-6, mab B and IT had no cytotoxic effect on HL-60. The IT showed a higher cytotoxic effect than SAP-6 in CEA-positive cell lines. The IT demonstrated the highest cytotoxic effect of 51% inhibition of control at 10(-7) M on the LS 174 T.

Hormonotoxins: synthesis, characterization and bioefficacy of some defined disulfide linked conjugates of ovine LH with a ribosome inactivating protein, gelonin.

In order to synthesise a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, gelonin, a single chain RIP obtained from an Indian plant, Gelonium multiflorum of Euphorbeaceae family was covalently linked to oLH with the use of N-succinimidyl-3-(2-pyridyldithio)propionate, generating a linkage containing a disulfide bond and a amide bond. The hormonotoxins were separated according to their molecular weight (indirectly according to oLH:gelonin molar ratio) and a complete biochemical analysis was performed. The linkage occurred through the epsilon-NH2 group of alpha oLH as judged from RP-HPLC analysis. The conjugates were devoid of ingredients as determined by SDS-PAGE and RP-HPLC analysis. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity of oLH and gelonin to their antibodies. Hormonotoxins bind to the Leydig tumour cells via oLH part leaving gelonin free as judged by competitive displacement analysis. The hormonotoxin was internalized to the sufficient degree to effectively inhibit protein synthesis. The cytotoxicity of 1:1 molar ratio conjugate was relatively higher than that of others. The cytotoxicity of presently described more defined hormonotoxins exhibited higher receptor binding and cytotoxicity than the hormonotoxins reported earlier [Singh, et al., J Biol Chem, 264 (1989) 3089].