Nutritional Evaluation of Milk Thistle Meal as a Protein Feedstuff for Diets of Dairy Cattle.

The objective of this work is to investigate the chemical and nutritional value of milk thistle meal (MTM) in order to improve it and to provide theoretical support for its application in dairy cattle production. MTM was assessed in comparison with seven conventional protein feed sources, namely, soybean meal (SBM), cottonseed meal (CS), canola meal (CN), palm kernel meal (PK), rice bran meal (RB), corn germ meal (CG), and sesame meal (SS). The chemical composition of these feedstuffs was assessed using wet chemical analysis, the Cornell Net Carbohydrate and Protein System was used to evaluate the carbohydrate and protein fractions, and the in situ nylon bag technique and the modified three-step in vitro method were used to assess the rumen degradation and intestinal digestibility. Additionally, Fourier transform infrared technology was used to determine the feedstuff protein spectral molecular structure and its amino acid profile was also assessed. The result showed that MTM acid detergent fiber, lignin, unavailable nitrogen, and non-degradable carbohydrate content were higher than those of the other feedstuffs. It had a 17% and 36% rumen effective degradation rate of neutral detergent fiber and dry matter, respectively, and had the lowest small intestinal rumen undegradable protein digestibility rate. It was low in leucine, histidine, arginine, and proline, but high in methionine. The total area of amide I and amide II in the protein secondary structure was similar to that of CN and CS, and the amide I and II ratio was not different from that of RB. To sum up, MTM has a poor carbohydrate composition and is high in fiber but, in comparison to most other protein feeds, has a higher crude protein rumen effective degradation rate, similar to that of SBM, and it is a good source of methionine, a limiting amino acid. Hence, its nutritional value can be further improved for application in dairy feeding through processes such as microbial or enzymatic fermentation.

Arginine-rich membrane-permeable peptides are seriously toxic.

The membrane-permeable peptides (MPP) such as undecapeptides TAT (YGRKKRRQRRR) and CTP (YGRRARRRRRR) have been receiving much attention for delivering various kinds of low membrane-permeability materials in vitro and in vivo. We have successfully used MPP in carrying various proteins through blood-brain barrier (BBB) in treatment of many kinds of nervous diseases. However, people always concentrate their mind on the efficacy and the mechanism of permeation of the conjugates across BBB, but overlook the toxicity of the membrane-permeable peptide itself. Once we injected intravenously not very large amounts of gamma-aminobutyric acid-MPP (GABA-MPP) to the mice, to our great surprise, the mice died within seconds with seizure, whereas the GABA control mice well survived. Thus, the importance of the toxicity of MPPs and their conjugates comes into the field of our vision. The low LD50 values of arginine-rich TAT (27.244 mg kg-1 ) and CTP (21.345 mg kg-1 ) per se in mice indicate that they all fall within the range of highly toxic chemicals. Among the arginine-rich peptides, R11 (RRRRRRRRRRR), a peptide composed purely of arginine residues, has the lowest LD50 value (16.5 mg kg-1 ) and manifests the highest toxicity, whereas TD (ACSSSPSKHCG), a peptide without arginine residue, shows a much lower toxicity and higher survival rate in mice. The mass percentage of arginine-rich MPP in the conjugate is critically important, the mass radio of arginine in the MPP appears a linear correlation with the toxicity. Thus we conclude, the arginine-rich MPPs are more suitable for using in the macro-molecular conjugates, but not in the small-molecular one.

Ilomastat, a synthetic inhibitor of MMPs, prevents lung injury induced by γ-ray irradiation in mice.

Lung injury is one of the pathological features in human or animal after radiation and the main side effect for patient after lung cancer radiotherapy. The efficient protective strategy still needs to exploit and the underlying mechanisms remain to be investigated. We found that the expression and activity of matrix metalloproteinases (MMPs) significantly increased at the early stage of radiation-induced lung injury (RILI). Pretreatment with Ilomastat, a synthetic inhibitor of MMPs, decreased the expression and activity of MMPs and significantly alleviated the lung inflammation and fibrosis in the irradiated mice, as well as enhanced the survival of irradiated mice. In addition, the levels of TGF-ß, IL-6, TNF-α and IL-1ß in the tissues dramatically reduced in the irradiated mice pretreated with Ilomastat. Furthermore, our experiments in vitro also showed that radiation significantly increased the MMPs activity, and Ilomastat pretreatment inhibited the activity of MMPs activated by irradiation and increased the cell survival. It is the first report, to our knowledge, to demonstrate that Ilomastat is a potential effective reliever for RILI and MMPs may play important roles in the process of RILI.

BBB: Permeable Conjugate of Exogenic GABA.

Neurotransmitters are the key factors in ameliorating the symptoms of nervous system diseases. Stroke/cerebral ischemia has been proven to be caused by the excess release of excitatory amino acid glutamate in the brain, and the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is considered to be the best choice to counteract the action of glutamate. Here, we show that GABA conjugated to a cytoplasmic transduction peptide (YGRRARRRRRR) by means of custom chemical synthesis could penetrate through the blood-brain barrier, increasing the GABA level in the plasma of rats and mice, which, as a result, display a state of calmness and somnolence.

Generation of Alzheimer's Disease Transgenic Zebrafish Expressing Human APP Mutation Under Control of Zebrafish appb Promotor.

BACKGROUND: Amyloid peptide precursor (APP) as the precursor protein of peptide betaamyloid (ß-amyloid, Aß), which is thought to play a central role in the pathogenesis of Alzheimer's disease (AD), also has an important effect on the development and progression of AD. Through knocking-in APP gene in animals, numerous transgenic AD models have been set up for the investigation of the mechanisms behind AD pathogenesis and the screening of anti-AD drugs. However, there are some limitations to these models and here is a need for such an AD model that is economic as well as has satisfactory genetic homology with human. METHODS: We generated a new AD transgenic model by knocking a mutant human APP gene (APPsw) in zebrafish with appb promoter of zebrafish to drive the expression of APPsw. RESULTS: Fluorescent image and immunochemistry stain showed and RT-PCR and western blot assay confirmed that APPsw was successfully expressed in the brain, heart, eyes and vasculature of the transgenic zebrafish. Behavioral observation demonstrated that the transgenic zebrafish had AD-like symptoms. Histopathological observation found that there were cerebral ß-amyloidosis and angiopathy (CAA), which induced neuron loss and enlarged pervascular space. CONCLUSION: These results suggest that APPsw transgenic zebrafish well simulate the pathological characters of AD and can be used as an economic AD transgenic model. Furthermore, the new model suggested that APP can express in microvasculatures and cause the Aß generation and deposition in cerebral vessel which further destroys cerebral vascular structure resulting in the development and/or the progress of AD.

Amelioration of dementia induced by Aß 22-35 through rectal delivery of undecapeptide-hEGF to mouse brain.

A group of growth factors have been shown to play important roles in amelioration of the malfunction of the neurodegenerative diseases. However, the proteins or polypeptides passing across the blood-brain barrier (BBB) to access the brain parenchyma are relatively few so that it hinders the therapies in clinic. Here a genetically reconstructed fusion peptide of human epidermal growth factor (hEGF) with an undecapeptide YGRKKRRQRRR (P11) was used to investigate the permeability between the cell membrane and the BBB via rectal administration. The efficiency to rescue the Aß 22-35-induced dementia in mice was assessed after administration of P11-hEGF per rectal. Our results showed that P11-hEGF permeates across not only the 3T3 cell membrane in vitro, but also the endothelia of vessels after intravenous injection (IV), and the mucosa of the rectum after per rectal administration. Further results showed that the circulating P11-hEGF allowed penetrating through the blood-brain barrier and then getting into the brain manifesting biological responses. In the animal experiments, treatment with P11-hEGF not only ameliorated the dementia induced by Aß 22-35 but also rescued the dementia of the aged mice, no matter how it was administrated (IV or per rectal). These results suggest that the rectal non-invasive delivery of the P11 polypeptide-conjugated growth factor is an efficient way for BBB transduction, thus raises the hope of real therapeutic progress against neurodegenerative diseases.

Transmembrane delivery and biological effect of human growth hormone via a phage displayed peptide in vivo and in vitro.

For a long time, people have been looking forward to being able to clinically deliver bio-drugs systemically by a noninvasive method. Here, we show that a synthetic peptide, TD (ACSSSPSKHCG) was efficient in transferring human growth hormone (GH) across various kinds of membranes and the blood-brain barrier (BBB) in vivo via rectal administration, resulting in elevation of GH level in serum, acetylcholine and O-choline acetyltransferase activities and GH /IGF-1 contents in brain tissues, manifesting great therapeutic effects on chronic age-related dementia in mice and ameliorating neuronal damage in the brain. Furthermore, the effects of Aß and TD/GH on LDH release, apoptosis and its relevant gene expression, involving bcl-2 and bax/caspase-3, were observed in a human neuroblastoma cell line (SH-SY5Y). Results indicated that GH decreased LDH release, apoptosis, and bax/caspase-3 activity, and increased bcl-2 expression compared with Aß treatment, moreover, TD/GH may enhance the effects due to existence of TD, which might be dependent on TD assisted cross-membrane delivery of GH. The transdermal/transmembrane-enhancing activity of the TD peptide was also manifested on porcine abdominal skin in vivo and the murine embryonic fibroblast cell line (3T3 cell) in vitro, which was further shown through interaction between TD and lecithin (one constituent of the cell membrane) by ESI-MS. In conclusion, TD/GH counteracted brain defects in aged mice in vivo and cell apoptosis induced by Aß in vitro might explain several underlying mechanisms by which GH could ameliorate learning and memory deficits in aged mice. Mixed TD/GH transmembrane delivery might be a promising therapy of Alzheimer's disease.

[PTD-hEGF fusion protein--gene expression and function analysis].

To produce membrane-permeable human epidermal growth factor (hEGF), a pPTD-hEGF prokaryocyte expression vector was constructed and transformed into E. coli BL 21 (DE3). The PTD-hEGF fusion protein was induced by 0.3 mmol/L of IPTG expressed in the form of inclusion body with an yield of 40% of the total protein in the cells, and then purified by Ni2+ -NTA affinity chromatography. The SDS-PAGE analysis showed a single fusion protein band with a molecular weight of 16 kD. The amino acid sequence was checked by MALDI-TOF-MS analysis. The genetic engineering PTD-hEGF fusion protein obviously promoted the proliferation and growth of the HEK-293 cells in vitro.

Transgenic mini-tomato and protection against alcohol-induced gastric injury.

The epidermal growth factor (EGF) has been shown to promote the proliferation of various types of cells, to maintain the physiological function of the mucosa of the digestive tract, and to promote the healing of the gastric and duodenal ulcers. It has been expressed in many types of bacteria and yeasts. In this article, a bio-reactor was constructed, namely, the human EGF (hEGF) transgenic mini-tomato. On the basis of hEGF gene sequence, a tomato codon preference hEGF gene was chemically synthesized, and it was constructed into the plant expression vector pCAMBIA2300. The transgenic tomato plants containing gene hEGF were obtained through Agrobacterium-mediated transformation. The expression product was determined by radioimmunoassay (RIA) and showed a yield of 3.48 +/- 1.01 ng/g fresh fruits. The intragastric gavage (ig) administration of the rhEGF-containing juice of the transgenic tomato (equivalent to 24 ng rhEGF per mouse a day) for 15 days could significantly protect mice against the alcohol-induced ulceration. The ulcer index, expressed as a degree of the stomach lesion, decreased from 42.20 +/- 18.13 to 16.25 +/- 9.57.

Protective effect of N-acetyl-L-cysteine on amyloid beta-peptide-induced learning and memory deficits in mice.

This study aimed to examine the effects of N-acetyl-L-cysteine (NAC) on protecting neurons function and improving learning and memory deficits in mice. Mice were intracerebroventricularly (icv) injected with the aggregated amyloid beta-peptide (Abeta) to produce Alzheimer's disease (AD). Learning and memory functions in mice were examined by the step through test and the water maze performance. The results showed that the mice pretreated with NAC had significantly greater retention in the step through test and shorter latencies in the water maze performance. Biochemical studies showed the potential role of free radical toxicity and the damage of cholinergic neurons in the Abeta-treated mice. There was an increased lipid peroxidation as indicated by elevated malondehyde (MDA) and decrease of glutathione (GSH) levels. There was also an increase in acetylcholinesterase (AChE) activity and a reduction in the choline acetyltransferase (ChAT) activity and acetylcholine (ACh) levels. NAC pretreatment significantly reversed the elevated MDA, AChE and the reduced GSH, ChAT and ACh in the Abeta-model mice. The results of the present study suggest the potential usage of the neuroprotective action of NAC on AD.

High-level secretion and purification of recombinant acetylcholinesterase from human cerebral tissue in P. pastoris and identification by chromogenic reaction.

The gene encoding human cerebral tissue acetylcholinesterase (AChE) was cloned from an 18-week fetal cerebral tissue and expressed in Pichia pastoris. Twenty-two positive transformants were obtained by Mut(+)/Mut(s) phenotypes screening in MD/MM medium and polymerase chain reaction amplification, and four recombinant P. pastoris strains that could secrete active AChE at high level were identified by simple and specific development reaction with indoxyl acetate as the chromogenic substrate. In shake-flask culture induced with methanol, the recombinant human AChE (rhAChE) content was about 76% of the total secreted proteins, and rhAChE activity in supernatant was 40 U/ml. The enzyme was purified through anion-exchange and affinity chromatography. Purity of the rhAChE was up to 96% after the simple purification procedure. The enzymatic activity reached 200 U/mg.

High-level expression of recombinant human paraoxonase 1 Q in silkworm larvae (Bombyx mori).

Human serum paraoxonase 1 (hPON1) belongs to a family of enzymes that catalyze the hydrolysis of a broad range of esters and lactones. Although the very first identification of hPON1 might have been as a calcium-dependent paraoxonase/arylesterase, PON1 is in fact a lactonase associated with high-density lipoprotein and strongly stimulated by apoA-I. PON1 hydrolyzes various organophosphates, including insecticides and nerve gases. PON1 also plays a key role in prevention of atherosclerosis. Mediation of cholesterol efflux from macrophage is a key in vivo function of PON1. In present study, the hPON1 Q gene was cloned into baculovirus transfer vector pVL1392 and expressed in silkworm expression system. The rhPON1 Q presented two bands with every near molecular weight of about 40 and 43 kDa according to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis. The expression level was up to 1,256 mg/L in haemolymph, about 50 times as high as that from BmN cells (24.8 mg/L). After purified by two chromatography steps (DEAE-Sepharose and HiTrap Chelating HP), the purity of rhPON1 Q was up to 90%, and the enzymatic properties are similar to serum hPON1.

Antisense inhibition of acetylcholinesterase gene expression for treating cognition deficit in Alzheimer's disease model mice.

To examine whether the selected antisense oligodeoxynucleotides (AS-ODN) targeting against human brain acetylcholinesterase (AChE) mRNA could improve the cognitive deficit in the Alzheimer's disease (AD) model mice induced by amyloid-beta peptide (Abeta), we determined the time-effect relationship of AChE activity and the learning and memory after AS-ODN delivery. The results showed that the AChE activity decreased gradually along with time, initiating at 8 h and lasting 42 h. The time-effect curves of acetylcholine (ACh) behaved consistency with that of AChE activity. The animal cognition studies showed that in step-through test, the error number of the AS-ODN-treated AD model mice was significantly decreased, and the memory retention was increased. In the water maze performance, the swimming time obviously shortened. Our results indicated that antisense therapy is of potential use in the treatment of cognitive deficit in the Abeta model mice.

Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.

A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.

Purification and characterization of recombinant human liver prolidase expressed in Saccharomyces cerevisiae.

The recombinant human liver prolidase (rh-prolidase, EC 3.4.13.9) from the lysate supernatant of engineering yeast Saccharomyces cerevisiae was purified in two steps employing anion-exchange gradient chromatography (DEAE-Sepharose fast flow) and gel filtration chromatography (Sephacryl S-200 high resolution). The purified recombinant protein furnished a single band with a molecular weight of 56 kD. Intensity scanning of the SDS-PAGE gel revealed that the prolidase accounted for more than 90% of total protein. The optimum pH of the catalytic reaction was 8.0. The enzyme was stimulated by Mn2+, but strongly inhibited by Cu2+ and Zn2+. The rh-prolidase expressed in S. cerevisiae had both dipeptidase and organophosphorus acid anhydrolase activity. It catalyzed the hydrolysis of soman and the dipeptide Gly -Pro. In a detoxification test in vitro, purified rh-prolidase was remarkably efficient at eliminating the toxicity of a lethal dose of soman, with the result that mice survived injection of such a dose.

Production of human liver prolidase by Saccharomyces cerevisiae as host cells.

AIM: To clone and express the recombinant human liver prolidase in yeast and explore the activities of both dipeptidase and organophosphoric acid anhydrolase (OPAA). METHODS: The cDNA encoding human liver prolidase derived from healthy adult liver was cloned into the pYES2, an expression vector of S cerevisiae, and then transformed into S cerevisiae INVSc1 by electroporation. The transformant with the highest enzymatic activity was induced by galactose for expression. The optimal induction conditions (temperature, induction time, and the initial amount of inoculation cells) were estimated by orthogonal experimental design. The recombinant prolidase and OPAA activities were assayed by spectrocolorimetric methods. RESULTS: The recombinant enzyme catalyzed the hydrolysis of organophosphorous compound soman as well as the hydrolysis of dipeptide Gly-Pro. Under the optimal induction conditions (20 h, 25 degree, initial OD(600)=0.4), the maximum activities of prolidase and OPAA came to 226.5 and 578 micromol.min(-1).g(-1) protein in cell lysate supernatants, respectively. SDS-PAGE of the recombinant enzyme in disrupted cell supernatants showed a molecular weight of 56 kDa. Intensity scanning of the SDS-PAGE gel revealed that the enzyme accounted for 3.16 % of the total protein in the supernatant. One liter incubation medium produced 7 g of wet yeast cell containing 4.56 mg of the recombination protein. CONCLUSION: The recombinant human liver prolidase produced by yeast cell (S cerevisiae) exhibited both dipeptidase and OPAA activities.

Anti-epitope antibody, a novel site-directed antibody against human acetylcholinesterase.

AIM: To construct synthetic antigens using the epitope of human brain acetylcholinesterase (hbAChE) for induction and detection of the specific antibody against the epitope, and to analyse the immunogenicity of the antibody. METHODS: The epitope (RTVLVSMNYR, amino acids 143-152) of hbAChE was chemically synthesized, coupled with the carrier protein keyhole limpet hemocyanin (KLH) to construct an artificial immunogen (KLH-epitope), and injected into rabbits to raise antibody. The epitope conjugated with bovine serum albumin (BSA) was used as the detection antigen. The specificity of the antibody was tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The immunoreaction between the anti-recombinant human butyrylcholinesterase (rhBChE) polyclonal antibody and the biotinylated-epitope was examined by indirect ELISA. RESULTS: The erythrocyte AChE, the hbAChE, rhBChE and the BSA-epitope all immunoreacted with the anti-epitope antibody against the epitope (143-152) of hbAChE, whereas the torpedo AChE did not. CONCLUSION: The hbAChE, the human erythrocyte AChE and hBChE share the conservative antigenic epitope RTVLVSMNYR, hence they can all immunoreact with the anti-epitope antibody. Since the epitope of hbAChE is less similar with the aligned amino acid sequences of AChE of Torpedo californica or Torpedo marmorata, there is not any immunoreactivity between them. The R, M, and N residues in the epitope seem to be necessary radicals for the conservation of antigenicity.

Screening of RNA molecules inhibiting human acetylcholinesterase by virtue of systematic evolution of ligands by exponential enrichment.

AIM: To acquire the specific RNA aptamers inhibiting human red blood cell (RBC) acetylcholinesterase (AChE). METHODS: Systematic evolution of ligands by exponential enrichment (SELEX) aptamer against human red blood cell membrane AChE was selected by microtiter plate method in vitro. The specifity binding to AChE was determined by gel mobility shift analysis. Microcolorispectrophotometric method was used to measure the activity of AChE. RESULTS: The aptamers to human RBC AChE were identified by 9 reiterative rounds. At the same concentration (2.25 micromol/L), the aptamers did not bind to the recombinant human butyrylcholinesterase (rhBChE) but specifically bound to human RBC AChE and inhibited the enzyme activity. CONCLUSION: It is an effective way to isolate the specific AChE inhibitor from the vast oligonucleotide combinatorial library by virtue of SELEX.

Molecular simulation of a single-chain antibody against AChE to explore molecular basis of inhibitory effect of 3F3 McAb on enzyme activity.

AIM: To explore the molecular basis of the inhibitory effect of 3F3, a monoclonal antibody against acetylcholinesterase (AChE), by computer-aided molecular simulation. METHODS: The single-chain 3F3 antibody (Sc3F3) was designed by joining VH and VL via a flexible linker (Gly4Ser)3. The amino acid sequence of the recombinant Sc3F3 was then subjected to computer-aided molecular modeling, and docking with the antigen molecule AChE to mimic the immunoactive interaction in a three-dimensional fashion. RESULTS: The modeled structure of Sc3F3 manifested the common features of a classical antibody. Both VH and VL were composed of two ?-sheets and connecting loops. The docking profile of the action between Sc3F3 with AChE demonstrated the formation of a stable structure. The van der Waals force played an important role suggesting that the complex was formed mainly via hydrophobic interactions between Sc3F3 and AChE molecules. CONCLUSION: The spatial structure of the complex of Sc3F3 and AChE showed that Sc3F3 overlaid the entrance of the active center gorge of AChE blocking the access of substrate.