Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines

The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest inductor of the immune response. There are several available commercial vaccines against bovine viral diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts. An estimated amount of 20 μg of tE2 per gram of fresh leaves was regularly obtained with this plant system. Injection of guinea pigs with plant extracts containing 20 μg of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines. This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the advantage to be free of any eventual animal contaminant.     

Supplementation with branched-chain amino acids attenuates hepatic apoptosis in rats with chronic liver disease

Branched-chain amino acids (BCAA) can function as pharmacologic nutrients for patients with decompensated cirrhosis. However, the effects of BCAA at the early stage of chronic liver disease are not clear. We hypothesized that early BCAA supplementation would attenuate the progression of chronic liver disease. The present study examined the effects of BCAA supplementation on the progression of chronic liver disease in rats caused by injected carbon tetrachloride (CCl₄). Sprague-Dawley rats were fed with a casein diet (control group) or the same diet supplemented with BCAA (BCAA group) for 11 weeks, and all rats were repeatedly injected with CCl₄. Food intake did not significantly differ between control and BCAA groups during the experimental period. Plasma alanine aminotransferase activities gradually increased during the experimental period in both groups but peaked later in the BCAA group. Liver fibrosis was more evident in the control group. Levels of connective tissue growth factor messenger RNA were significantly lower in the livers of rats in the BCAA group than in the control group. Terminal deoxynucleotidyl transferase–mediated deoxyuridine 5-triphosphate nick end labeling assays found considerably more hepatic apoptosis in the control group. Liver cytosolic cytochrome c levels and expression of the proapoptotic Bax protein in the mitochondrial fraction were significantly lower in the BCAA group than in the control group. These results suggest that supplementation with BCAA delays the progression of chronic liver disease caused by CCl₄ in rats by attenuating hepatic apoptosis.     

Thyroid hormone improves the mechanical performance of the post-infarcted diabetic myocardium: A response associated with up-regulation of Akt/mTOR and AMPK activation

Objective

Thyroid hormone (TH) is shown to be protective against cardiac and pancreatic injury. Thus, this study explored the potential effects of TH treatment on the functional status of the postinfarcted diabetic myocardium. Diabetic patients have worse prognosis after acute myocardial infarction (AMI).

Materials/Methods

AMI was induced by left coronary ligation in rats previously treated with 35 mg/kg streptozotocin (STZ), (DM-AMI). TH treatment was initiated at 2 weeks after AMI and continued for 6 weeks (DM-AMI + TH), while sham-operated animals served as control (DM-SHAM).

Results

TH treatment increased cardiac mass, improved wall stress and favorably changed cardiac geometry. TH significantly increased echocardiographic left ventricular ejection fraction (LVEF%): [54.2 (6.5) for DM-AMI + TH vs 37 (2.0) for DM-AMI, p < 0.05]. TH treatment resulted in significantly increased insulin and decreased glucose levels in serum. The ratios of phosphorylated (p)-Akt/total Akt and p-mTOR/total mTOR were increased 2.0 fold and 2.7 fold in DM-AMI + TH vs DM-AMI respectively, p < 0.05. Furthermore, the ratio of p-AMPK/total AMPK was found to be increased 1.6 fold in DM-AMI + TH vs DM-AMI, p < 0.05.

Conclusion

TH treatment improved the mechanical performance of the post-infarcted myocardium in rats with STZ-induced diabetes, an effect which was associated with Akt/mTOR and AMPK activation.     

A serine proteinase in the penetration glands of the hexacanths of Hymenolepis diminuta (Cestoda, Cyclophyllidea)

 Histochemically demonstrable activity of a serine proteinase was detected in the penetration glands of Hymenolepis diminuta hexacanths. At the optimal pH of 8.4 the enzyme hydrolyzed N-blocked l-aminoacyl- and N-blocked l-peptidyl-naphthylamides bearing l-arginine at the P₁ subsite. The proteinase did not require either Ca²⁺ or Mg²⁺ for its activity and was insensitive to 1 mM EGTA and 1 mM EDTA. Organic fluorophosphates inhibited it, whereas thiol-blocking compounds did not. At operative pH values of 4.8 and 3.8 generated during electrophoresis in a stacking and a resolving gel, respectively, the proteinase migrated toward the cathode. When examined for proteolytic activity at the optimal pH of 8.4, the separated enzyme produced a single band of gelatinolysis in a gelatin-containing polyacrylamide gel. During in vitro maintenance of the hexacanths, the secretion from their penetration glands formed a mucous cyst surrounding the individual larvae. The cyst was resistant to and protected the hexacanths from the proteolytic activity of trypsin, papain, and proteinases extracted from the gut of the beetle Tenebrio molitor (the host). Hexacanths extracted from the hemocoel of T. molitor at 24 and 48 h after infection were surrounded by similar mucous cysts. Consequently, roles in penetration and protection for the secretion from the penetration glands are postulated. Received: 18 January 1995 / Accepted: 20 May 1995     

Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14)

In the present paper, the distribution of a neuron-specific phosphoneuroprotein 14 (PNP 14) in cell and tissue was investigated in detail by the immunoblot method using affinity-purified antibody against this protein. The immunoblot of the supernatant fractions of various tissue homogenates of rat clearly demonstrated that PNP 14 was enormously rich in the brain. The content in rat brain was as much as 0.1% of the homogenate. The immunocytochemical study showed that the protein was localized at nerve endings in the cerebellum. Existence of the protein was also comfirmed in cultured neuronal cells from postnatal rat midbrain, but not in glial cells. Examination of subcellular localization of PNP 14 indicates that the protein was present in synaptic plasma membranes and synaptic supernatant fractions, but not in synaptic vesicles. During the development of rat brain, PNP 14 came into existence after birth and it's amount linearly increased to a maximum at 21–28 days after birth. The content of the protein then remained at the same level for more than 10 months. We concluded that this protein is neuron specific and supposed that it may be involved in neuronal formation and function.     

Immunopurification of human plasma kininogens

Human plasma kininogens were purified by immunoadsorption on Sepharose columns using two different approaches, either removing protein impurities with the respective immunospecific polymers or applying an anti-kininogen-specific immunoadsorbent column. An anti-kininogen serum developed and investigated in this laboratory in earlier studies was used. This antiserum recognizes the native conformational determinants in the kininogen heavy chain, the common denominator in plasma kininogens, and reacts with three heterogeneous molecular forms of high mol. wt kininogen (mol. wts 103,000, 92,000 and 90,000) as well as with low mol. wt kininogen. Heterogeneity of kininogens was shown by SDS gel electrophoresis and immunoelectrophoresis. With the antibody-specific polymers the yield was 80-100% compared to 75% or lower when several consecutive immunoadsorption steps were applied to remove impurities. Both methods serve the purpose of preparing immunologically pure kininogens suitable for immunization.     

Changes in serum and exudate levels of functional macroglobulins and anti-inflammatory effect of alpha 2-acute-phase macroglobulin on carrageenin-induced inflammation in rats

Serum and exudate levels of functional macroglobulins that have the ability to inhibit proteinases were determined at various times after carrageenin injection into a preformed air-pouch on the back of rats. The trypsin-inhibiting activity of serum macroglobulins increased after a lag period of 3 hr, reached a maximum at 24 hr, and decreased steadily until day 16 after carrageenin injection. This change was in good agreement with the change in the serum level of alpha 2-acute-phase macroglobulin. In contrast with the serum level, the exudate level of functional macroglobulins was negligible on day 1, detectable on day 3, and remained at almost the same level from day 5 to day 16 after carregeenin injection. Macroglobulins were partially purified from rat serum obtained at 20 hr after carregeenin injection, and their anti-inflammatory activity was studied. The partially purified alpha 2-acute-phase macroglobulin and the alpha 1 macroglobulin were injected into the air-pouch immediately after carrageenin injection, with the result that a single injection of the functionally active alpha 2-acute-phase macroglobulin significantly inhibited the formation of granulation tissue on day 4 after the carrageenin injection, whereas functionally inactive alpha 1 macroglobulin was without effect. These results suggest that the inhibitory activity of macroglobulins on the development of granulation tissue is due to the proteinase-inhibiting capacity of macroglobulins.     

Isolation and characterization of the third and fifth components of rabbit complement

Highly purified rabbit C3 and C5 have been obtained from normal rabbit plasma by differential precipitation and column chromatography. Apparent molecular weights of the two proteins as determined by SDS-polyacrylamide gel electrophoresis were 176,500 (C3) and 171,000 (C5), whereas reduction of the samples prior to electrophoresis allowed estimation of the molecular weights of the α and β subunit chains of each protein at 123,000 and 70,100 for C3 and 128,500 and 88,000 for C5. Serum concentrations as determined by rocket immunoelectrophoresis were 1.2 mg/ml for C3 and 0.2 mg/ml for C5, whereas hemolytic activities of C3 and C5 in normal rabbit serum were 2.4 × 10¹⁰ and 3.2 × 10¹⁰ effective molecules per ml, respectively. Amino acid analyses of the C3 and C5 preparations indicated similar but not identical amino acid compositions of the the two rabbit proteins, but there were no unusual amino acids or other features of the composition. Monospecific antisera to each of the two complement components were obtained in goats by immunization with the purified material.     

Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor Sepharose chromatography

Cilostamide derivatives are potent inhibitors of human platelet aggregation and selectively inhibit human platelet cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase. N-Cyclohexyl-N-(2-hydroxybutyl)-5-[6-1,2,3,4-tetrahydro-2-oxoquinolyl oxy)] -butyramide (OPC-13135) is one of these derivatives, and the concentration of OPC-13135 producing 50% inhibition of human platelet aggregation induced by 2 micrograms/ml collagen was 5 microM. On the other hand, the concentrations of OPC-13135 producing 50% inhibition of human platelet cyclic AMP phosphodiesterase and cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase were 0.073 and 21.8 microM, respectively. We purified over 480-fold the soluble low Km form of cyclic AMP phosphodiesterase from human platelets, using OPC-13135 Sepharose column as a final step in the purification procedure. The purified protein has a molecular weight of 175,000, determined by gel filtration and is an acidic protein, as determined by isoelectric focussing (pI = 4.9). Kinetic measurements indicated that the enzyme protein had a Km value for the substrate cyclic AMP and cyclic GMP of 0.34 and 0.11 microM respectively, and a Vmax value of 85.3 and 19.8 nmole/min/mg protein, respectively. Ki value of the OPC-13135 for the enzyme was 0.015 microM and was of competitive fashion against cyclic AMP.     

L-amino acid oxidase from Vipera lebetina venom: isolation, characterization, effects on platelets and bacteria.

The L-amino acid oxidase from Vipera lebetina venom was purified to homogeneity using combination of size exclusion, ion exchange and hydrophobic chromatography. The monomeric molecular mass of the homodimeric enzyme is 60.9kDa. The N-terminal and the tryptic peptides share high homology with other snake venom L-amino acid oxidases. The enzyme displays high specificity towards hydrophobic L-amino acids, the best substrates are L-Met, L-Trp, L-Leu followed by L-His, L-Phe, L-Arg and L-Ile. Six substrates-Gly, L-Ser, L-Thr, L-Pro, L-Cys, L-Asp--were not oxidized. The enzyme has antimicrobial activity inhibiting the growth of both Gram-negative and Gram-positive bacteria. V. lebetina LAAO dose-dependently inhibited platelet aggregation induced by ADP or collagen. In case of ADP-induced aggregation the inhibitory effect was more pronounced on the second wave of aggregation.