Cloning,Expression, and Characterization of Rice α-Galactosidase

We have successfully cloned an α-galactosidase gene from a rice cDNA library and transformed it into Escherichia coli BL21. It was subsequently cloned to the pPIC9K vector and expressed in Pichia pastoris. A selected clone was found to result in high production yield of the galactosidase enzyme. The secreted enzyme was purified, and it revealed as a major protein band on an SDS-PAGE gel. The optimal pH value, enzyme stabilities, and substrate specificity were studied. The enzyme specificity toward the terminal α1→6, 1→4, and 1→3 linked galactosyl residue from various substrates was investigated. By determining the Michelis constant (Km) of the enzyme for melibiose, raffinose, and stachyose, our results showed that melibiose was hydrolyzed faster than raffinose, whereas the published data reported a reversed sequence, raffinose > melibiose. The enzyme also showed the ability of converting B red blood cells into O red cells. The objective of this work is to develop the Pichia system to produce a large quantity of enzyme for blood cell conversion for transfusion.     

Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm–Baculovirus Expression System

Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm–BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm–BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans.     

Expression of core-binding factor α1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts

To study the effects and importance of fluoride on FBs in the development of extraperiosteal calcification and the ossification of skeletal fluorosis, the presence of the osteogenic phenotype, which is indicated by the expression of core-binding factor α1 (Cbfa1) and osteocalcin (OCN), in an FB cell line (L929) and in osteoblasts (OBs) exposed to fluoride was determined. Fibroblasts and osteoblasts were exposed to different concentrations of fluoride (0, 0.0001, 0.001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻). By using RT-PCR and ELISA, the mRNA levels of Cbfa1 and OCN were measured at 48 h, and the protein levels of Cbfa1 and OCN were measured at 2, 4, 24, 48 and 72 h. The data demonstrated the following: (1) The Cbfa1 protein level in fluoride-treated fibroblasts clearly increased at 48 h in the groups treated with 0.0001, 0.001, 0.1, 1.0 and 20.0 mg/L F⁻. The Cbfa1 protein level of the group treated with 10 mg/L F⁻ at 72 h was higher than that of the control group. The level of Cbfa1 mRNA in the fibroblasts was much higher at 48 h in the group treated with 10.0 mg/L F⁻ than in the control group. (2) The OCN protein level in fluoride-treated fibroblasts was significantly higher than that of the control group in the 0.0001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻ groups at 2 h, and in the 0.001 and 0.1 F⁻ groups at 4 h. A slightly higher level of OCN mRNA in fluoride-treated fibroblasts was also found in the 1.0 and 20.0 mg/L F⁻ groups compared to the control group. (3) The expressions of Cbfa1 and OCN in osteoblasts treated with the same experimental conditions as the fibroblasts were up-regulated by fluoride following the same trend as in the fibroblasts. Our results showed an increase in the expression of Cbfa1 and OCN in fibroblasts and osteoblasts exposed to fluoride and suggested that the osteogenic function of fibroblasts induced by fluoride could play an important role in the development of extraperiosteal ossification during skeletal fluorosis.     

VEGF,HIF-1α Expression and MVD as an Angiogenic Network in Familial Breast Cancer

Angiogenesis, which plays an important role in tumor growth and progression of breast cancer, is regulated by a balance between pro- and anti-angiogenic factors. Expression of vascular endothelial growth factor (VEGF) is up-regulated during hypoxia by hypoxia-inducible factor-1α (HIF-1α). It is known that there is an interaction between HIF-1α and BRCA1 carrier cancers, but little has been reported about angiogenesis in BRCA1-2 carrier and BRCAX breast cancers. In this study, we investigated the expression of VEGF and HIF-1α and microvessel density (MVD) in 26 BRCA1-2 carriers and 58 BRCAX compared to 77 sporadic breast cancers, by immunohistochemistry. VEGF expression in BRCA1-2 carriers was higher than in BRCAX cancer tissues (p = 0.0001). Furthermore, VEGF expression was higher in both BRCA1-2 carriers and BRCAX than the sporadic group (p<0.0001). VEGF immunoreactivity was correlated with poor tumor grade (p = 0.0074), hormone receptors negativity (p = 0.0206, p = 0.0002 respectively), and MIB-1-labeling index (p = 0.0044) in familial cancers (BRCA1-2 and BRCAX). The percentage of nuclear HIF-1α expression was higher in the BRCA1-2 carriers than in BRCAX cancers (p<0.05), and in all familial than in sporadic tumor tissues (p = 0.0045). A higher MVD was observed in BRCA1-2 carrier than in BRCAX and sporadic cancer tissues (p = 0.002, p = 0.0001 respectively), and in all familial tumors than in sporadic tumors (p = 0.01). MVD was positively related to HIF-1α expression in BRCA1-2 carriers (r = 0.521, p = 0.006), and, in particular, we observed a highly significant correlation in the familial group (r = 0.421, p<0.0001). Our findings suggest that angiogenesis plays a crucial role in BRCA1-2 carrier breast cancers. Prospective studies in larger BRCA1-2 carrier series are needed to improve the best therapeutic strategies for this subgroup of breast cancer patients.     

Carvedilol Decrease IL-1β and TNF-α, Inhibits MMP-2, MMP-9, COX-2, and RANKL Expression,and Up-Regulates OPG in a Rat Model of Periodontitis

Periodontal diseases are initiated primarily by Gram-negative, tooth-associated microbial biofilms that elicit a host response that causes osseous and soft tissue destruction. Carvedilol is a β-blocker used as a multifunctional neurohormonal antagonist that has been shown to act not only as an anti-oxidant but also as an anti-inflammatory drug. This study evaluated whether Carvedilol exerted a protective role against ligature-induced periodontitis in a rat model and defined how Carvedilol affected metalloproteinases and RANKL/RANK/OPG expression in the context of bone remodeling. Rats were randomly divided into 5 groups (n = 10/group): (1) non-ligated (NL), (2) ligature-only (LO), and (3) ligature plus Carvedilol (1, 5 or 10 mg/kg daily for 10 days). Periodontal tissue was analyzed for histopathlogy and using immunohistochemical analysis characterized the expression profiles of MMP-2, MMP-9, COX-2, and RANKL/RANK/OPG and determined the presence of IL-1β, IL-10 and TNF-α, myeloperoxidase (MPO), malonaldehyde (MDA) and, glutathione (GSH). MPO activity in the group with periodontal disease was significantly increased compared to the control group (p<0.05). Rats treated with 10 mg/kg Carvedilol presented with significantly reduced MPO and MDA concentrations (p<0.05) in addition to presenting with reduced levels of the pro-inflammatory cytokines IL-1 β and TNF-α (p<0.05). IL-10 levels in Carvedilol-treated rats remained unaltered. Immunohistochemical analysis demonstrated reduced expression of MMP-2, MMP-9, RANK, RANKL, COX-2, and OPG in rats treated with 10 mg/kg Carvedilol. This study demonstrated that Carvedilol affected bone formation/destruction and anti-inflammatory activity in a rat model of periodontitis.     

Expression of GFRα-1, GFRα-2, and c-Ret mRNAs in rat adrenal gland

Glial cell line-derived neurotrophic factor (GDNF), an important factor for developing and lesioned pre- and postganglionic sympathetic neurons, and its congeners signal through a receptor complex consisting of the tyrosine kinase c-Ret and a lipid-anchored α receptor (GFRα-1-4). Using in situ hybridization we show now that the mRNA for GFRα-2 is abundant in the adult rat adrenal medulla and its chromaffin cells. Coexpression of c-Ret and GFRα-1 mRNA's is restricted to a scarce subpopulation of medullary sympathetic neurons. Both GFRα-1 and GFRα-2 mRNA's are associated with preganglionic nerve trunks in the adrenal cortex. It is conceivable therefore that GDNF and related factors may activate chromaffin and preganglionic Schwann cells through a GFR-α receptor in absence of c-Ret.     

Expression, purification, and immunogenic characterization of Epstein–Barr virus recombinant EBNA1 protein in Pichia pastoris

Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390–641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4⁺ and CD8⁺ T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.     

Expression,Purification, and Characterization of Recombinant Human α<Subscript>1</Subscript>-Antitrypsin Produced Using Silkworm–Baculovirus Expression System

Human α₁-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α₁-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~?15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.     

Expression and secretion of β-glucuronidase and Pertussis toxin S1 by Streptomyces lividans

 Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coliβ-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the β-glucuronidase gene was fused with the leader sequence produced up to 30 mg β-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/l. The disappearence of the B. pertussis toxin S1 and β-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans. Received: 19 July 1995/Received revision: 3 November 1995/Accepted: 20 November 1995     

Zonula Occludens-1, Occludin and E-cadherin Expression and Organization in Salivary Glands with Sj?gren’s Syndrome

Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disorder that causes secretory dysfunction of the salivary glands leading to dry mouth. Previous studies reported that tight junction (TJ) proteins are down-regulated and lose polarity in human minor salivary glands with SS, suggesting that TJ structure is compromised in SS patients. In this paper, we utilized the NOD/ShiLtJ mouse with the main goal of evaluating this model for future TJ research. We found that the organization of apical proteins in areas proximal and distal to lymphocytic infiltration remained intact in mouse and human salivary glands with SS. These areas looked comparable to control glands (i.e., with no lymphocytic infiltration). TJ staining was absent in areas of lymphocytic infiltration coinciding with the loss of salivary epithelium. Gene expression studies show that most TJs are not significantly altered in 20-week-old NOD/ShiLtJ mice as compared with age-matched C57BL/6 controls. Protein expression studies revealed that the TJ proteins, zonula occludens-1 (ZO-1), occludin, claudin-12, as well as E-cadherin, do not significantly change in NOD/ShiLtJ mice. Our results suggest that ZO-1, occludin and E-cadherin are not altered in areas without lymphocytic infiltration. However, future studies will be necessary to test the functional aspect of these results.     

Expression of connexin 37, 40, and 43 mRNA and protein in renal preglomerular arterioles

Gap junctions allow direct intercellular coupling between many cells including those in the vascular wall. Studies of connexin expression in cells of the microcirculatory system are very few in number. However, cell-to-cell communication between cells of the arteriolar wall may be particularly important in microcirculatory control. We investigated the expression of connexins 43, 40, and 37 (Cx43, Cx40, Cx37) mRNA and proteins in primary cultures of smooth muscle cells (SMC) from rat renal preglomerular arterioles and in the aortic cell line A7r5. Furthermore protein expression in preglomerular arterioles in frozen sections was evaluated. SMC were isolated from kidneys using an iron oxide sieve method and explant technique. Total RNA from these cultures was tested by RT-PCR analysis for the expression of the three connexins mRNA. Using immunofluorescence we examined whether the expression pattern of connexin protein in the cell culture and frozen sections corresponded to the mRNA expression. The data show that A7r5 and preglomerular SMC express mRNA for Cx37 in addition to Cx43 and Cx40. In A7r5 cells the mRNA for Cx43, Cx40, and Cx37 are translated to protein, whereas cultured preglomerular SMC and the media of afferent arterioles in frozen sections only showed Cx40 immunoreactivity.     

Expression, purification, and refolding of active human and mouse secreted group IIE phospholipase A₂

Secreted phospholipase A?s form a large family of proteins involved in diverse biological and pathophysiological processes. Group IIE secreted phospholipase A? (sPLA?-IIE) is one of the latest discovered members of this family. Previous studies revealed that the expression profile of sPLA?-IIE was restricted to a few tissue types including brain, heart, lung and placenta compared to the broad expression profile of other isoforms. Accumulating evidence suggests that sPLA?-IIE might play a pivotal role in the progression of inflammatory processes. However, functional study of sPLA?-IIE was hindered by the low yield of soluble expressed protein. In this study, we have expressed human and mouse sPLA?-IIE in Escherichia coli in the form of inclusion bodies. The inclusion bodies were dissolved, purified and refolded in a step-wise dialysis approach and further purified. We obtained soluble and active proteins for human and mouse sPLA?-IIE with a final yield of 1.1 and 1.2 mg/500 mL bacterial culture, respectively. The refolded sPLA?-IIEs exhibited similar calcium and pH dependence of their enzymatic activity with those expressed in COS cells. This protein expression and purification protocol will facilitate the further structural and functional studies of human and mouse sPLA?-IIEs.     

Prokaryotic Expression and In Vitro Functional Analysis of IL-1β and MCP-1 from Guinea Pig

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.     

The Tolerogenic Peptide,hCDR1, Down-Regulates the Expression of Interferon-α in Murine and Human Systemic Lupus Erythematosus

Background

The tolerogenic peptide, hCDR1, ameliorated manifestations of systemic lupus erythematosus (SLE) via the immunomodulation of pro-inflammatory and immunosuppressive cytokines and the induction of regulatory T cells. Because type I interferon (IFN-α) has been implicated to play a role in SLE pathogenesis, we investigated the effects of hCDR1 on IFN-α in a murine model of SLE and in human lupus.

Methodology/Principal Findings

(NZBxNZW)F1 mice with established SLE were treated with hCDR1 (10 weekly injections). Splenocytes were obtained for gene expression studies by real-time RT-PCR. hCDR1 down-regulated significantly IFN-α gene expression (73% inhibition compared to vehicle treated mice, p = 0.002) in association with diminished clinical manifestations. Further, hCDR1 reduced, in vitro, IFN-α gene expression in peripheral blood mononuclear cells (PBMC) of 10 lupus patients (74% inhibition compared to medium, p = 0.002) but had no significant effects on the expression levels of IFN-α in PBMC of primary anti-phospholipid syndrome patients or of healthy controls. Lupus patients were treated for 24 weeks with hCDR1 (5) or placebo (4) by weekly subcutaneous injections. Blood samples collected, before and after treatment, were frozen until mRNA isolation. A significant reduction in IFN-α was determined in hCDR1 treated patients (64.4% inhibition compared to pretreatment expression levels, p = 0.015). No inhibition was observed in the placebo treated patients. In agreement, treatment with hCDR1 resulted in a significant decrease of disease activity. IFN-α appears to play a role in the mechanism of action of hCDR1 since recombinant IFN-α diminished the immunomodulating effects of hCDR1 on IL-1β, TGFβ and FoxP3 gene expression.

Conclusions/Significance

We reported previously that hCDR1 affected various cell types and immune pathways in correlation to disease amelioration. The present studies demonstrate that hCDR1 is also capable of down-regulating significantly (and specifically to lupus) IFN-α gene expression. Thus, hCDR1 has a potential role as a novel, disease specific treatment for lupus.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.