登革病毒包膜蛋白的原核表达及免疫原性研究

登革病毒感染引起的登革热和登革出血热/登革休克综合征是目前流行最为广泛的虫媒病毒病,但其发病机制不明,也无疫苗和特异性抗病毒药物用于防治。登革病毒包膜蛋白(E蛋白)在病毒致病和免疫过程中发挥重要作用。本研究构建了登革病毒2型E蛋白基因的重组质粒E/pGEX-6P-1,并优化表达条件,获得登革病毒E蛋白的高效可溶性表达;用谷胱甘肽琼脂糖凝胶4B亲和层析柱纯化,获得纯度较高的蛋白。该蛋白具有较好的免疫原性,免疫小鼠后可获得高效价抗体。本研究为进一步了解登革病毒E蛋白的功能及其在相关疾病中的应用奠定了基础。     

In vitro and in vivo interactions between the hepatitis B virus protein P22 and the cellular protein gC1qR

gC1qR, a mitochondrial matrix protein, was identified as the main cellular partner of the hepatitis B virus P22 protein. We demonstrated by immunofluorescence studies that some P22 molecules were colocalized with the endogenous gC1qR in both the cytoplasm and the nucleus but never in the mitochondria. We also showed that the last 34 amino acids of P22 were involved in the association with gC1qR.     

In vitro and in vivo assessment of herb drug interactions

Herbal products contain several chemicals that are metabolized by phase 1 and phase 2 pathways and also serve as substrates for certain transporters. Due to their interaction with these enzymes and transporters there is a potential for alteration in the activity of drug metabolizing enzymes and transporters in presence of herbal components. Induction and inhibition of drug metabolizing enzymes and transporters by herbal component has been documented in several in vitro studies. While these studies offer a system to determine the potential for a herbal component to alter the pharmacokinetics of a drug, they cannot always be used to predict the magnitude of any potential effect in vivo. In vivo studies are the ultimate way to determine the clinical importance of herb drug interactions. However, lack of content uniformity and lack of documentation of the bioavailability of herbal components makes even in vivo human studies difficult to interpret as the effect may be product specific. It appears that St. John's wort extract is probably one of the most important herbal product that increases the metabolism and decreases the efficacy of several drugs. Milk thistle on the other hand appears to have minimal effect on phase 1 pathways and limited data exists for phase 2 pathways and transporter activity in vivo. Further systematic studies are necessary to assess the significance of herb drug interactions.     

In vitro and in vivo enzyme studies of polyhemoglobin-tyrosinase

Melanoma is now the fifth most common type of cancer in North America. At present, there is no optimal treatment for this cancer. However, the lowering of the tyrosine level can inhibit the growth of melanoma. Unfortunately, this diet restriction cannot be humanly tolerated and causes vomiting, nausea, and severe body weight loss. To prevent these problems, we are studying a new approach involving the preparation intermolecularly crosslinked hemoglobin and tyrosinase for intravenous injection. In this article we describe the method of preparation and the structural and functional properties of polyhemoglobin-tyrosinase. We evaluate the effects of varying glutaraldehyde ratio, crosslinking time, and enzyme concentration on the enzyme activity of polyhemoglobin-tyrosinase. We also optimize the molecular weight distribution of polyhemoglobin-tyrosinase. The stability of polyhemoglobin-tyrosinase at 37 degrees C is much more stable when compared to noncrosslinked tyrosinase solution. Animal studies show that a higher degree of polymerization correlates with a longer circulation time of polyhemoglobin-tyrosinase, and the optimal crosslinking time is 24 hours. One intravenous injection of polyhemoglobin-tyrosinase lowers the plasma tyrosine to about 10% of its original level within one hour.     

In vitro and in vivo studies of ICE inhibitors

Interleukin-1β-converting enzyme (ICE) is a cysteine protease responsible for proteolytic activation of the biologically inactive interleukin-1β precursor to the proinflammatory cytokine. ICE and homologous proteases also appear to mediate intracellular protein degradation during programmed cell death. Inhibition of ICE is a new antiinflammatory strategy being explored by the design of both reversible inhibitors and irreversible inactivators of the enzyme. Such compounds are capable of blocking release of interleukin-1β from human monocytes. ICE inhibitors that cross react against multiple ICE homologs can also block apoptosis in diverse cell types. ICE inhibitors impart protection in vivo from endotoxin-induced sepsis and collagen-induced polyarthritis in rodent models. Further optimization of the current generation of peptidyl ICE inhibitors will be required to produce agents suitable for administration in chronic inflammatory and neurodegenerative diseases. J. Cell. Biochem. 64:19–26. © Wiley-Liss, Inc.     

Castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo

Previous studies have suggested that α-glucosidase inhibitors such as castanospermine and deoxynojirimycin inhibit dengue virus type 1 infection by disrupting the folding of the structural proteins prM and E, a step crucial to viral secretion. We extend these studies by evaluating the inhibitory activity of castanospermine against a panel of clinically important flaviviruses including all four serotypes of dengue virus, yellow fever virus, and West Nile virus. Using in vitro assays we demonstrated that infections by all serotypes of dengue virus were inhibited by castanospermine. In contrast, yellow fever virus and West Nile virus were partially and almost completely resistant to the effects of the drug, respectively. Castanospermine inhibited dengue virus infection at the level of secretion and infectivity of viral particles. Importantly, castanospermine prevented mortality in a mouse model of dengue virus infection, with doses of 10, 50, and 250 mg/kg of body weight per day being highly effective at promoting survival (P ≤ 0.0001). Correspondingly, castanospermine had no adverse or protective effect on West Nile virus mortality in an analogous mouse model. Overall, our data suggest that castanospermine has a strong antiviral effect on dengue virus infection and warrants further development as a possible treatment in humans.     

In vitro and in vivo studies with adriamycin liposomes

Liposome entrapped adriamycin retains its full cytotoxic potential when tested under $\text{in}\text{vitro}$ conditions against murine leukemia L-1210 cells. $\text{In}\text{vivo}$ drug distribution studies indicate that, relative to the free drug, a lower proportion of adriamycin administered in the liposome form is delivered to the heart and kidneys at one and four hours after injection. When administered to normal mice in high doses, anionic adriamycin liposomes appear less harmful than equal doses of the free drug as judged by alterations in animal weight gain. In these studies, a noval double packing procedure has been used for the entrapment of adriamycin in phospholipid vesicles.     

In vitro and in vivo neurotoxicity of prion protein oligomers

The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.     

New methodologies for measuring protein interactions in vivo and in vitro

The identification and characterization of protein interactions is a key topic in current life science research; a huge variety of methodologies have been established in recent years to expedite research in this area. Generic methods have been established for monitoring protein interactions in vivo by protein fragment complementation and for screening protein interactions in vitro by highly parallel solid-phase techniques. Substantial progress has been made in identifying and characterizing interactions with and between membrane proteins. Studying protein interactions on the single-molecule level has become an important tool for understanding protein function in vivo and in vitro.     

In vitro and in vivo interactions of homocysteine with human plasma transthyretin

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and an emerging risk factor for cognitive dysfunction and Alzheimer's disease. Greater than 70% of the homocysteine in plasma is disulfide-bonded to protein cysteine residues. The identity and functional consequences of protein homocysteinylation are just now emerging. The amyloidogenic protein transthyretin (prealbumin), as we now report, undergoes homocysteinylation at its single cysteine residue (Cys10) both in vitro and in vivo. Thus, when human plasma or highly purified transthyretin was incubated with 35S-L-homocysteine followed by SDS-PAGE and PhosphorImaging, two bands corresponding to transthyretin dimer and tetramer were observed. Treatment of the labeled samples with beta-mercaptoethanol prior to SDS-PAGE removed the disulfide-bound homocysteine. Transthyretin-Cys10-S-S-homocysteine was then identified in vivo in plasma from normal donors, patients with end-stage renal disease, and homocystinurics by immunoprecipitation and high performance liquid chromatography/electrospray mass spectrometry. The ratios of transthyretin-Cys10-S-S-homocysteine and transthyretin-Cys10-S-S-sulfonate to that of unmodified transthyretin increased with increasing homocysteine plasma concentrations, whereas the ratio of transthyretin-Cys10-S-S-cysteine to that of unmodified transthyretin decreased. The hyperhomocysteinemic burden is thus reflected in the plasma levels of transthyretin-Cys10-S-S-homocysteine, which in turn may contribute to the pathological consequences of amyloid disease.     

In vitro and in vivo studies of antiosteosarcoma activities of formononetin

Osteogenic sarcoma (OGS) is a primary bone cancer, characterized by aggressive neoplasm from mesenchymal oncogenesis. However, the clinical therapeutic regimen against OGS is limited. Therefore, potential medication warrants to be further developed. Our previous study indicates that formononetin (FN) exerts effective pharmacological activity against OGS. This study aimed to further decipher the molecular mechanism behind this benefit. Patients with OGS were recruited for clinical data assay and immunoassay. Human OGS cell line (U2OS) and tumor-bearing nude mice were subjected to a battery of biochemical analyses and immunoassays for integrative evaluation of FN-exerted anti-OGS effects. In human data, OGS samples showed increased expressions of ERα, p-PI3KCA^Tyr317, and p-AKT ^Ser473 proteins, followed by notably upregulated miR-375 content in comparison with that in OGS-free. In addition, FN-treated U2OS cells showed inhibited cell proliferation, elevated lactic dehydrogenase production and lowered endogenous miR-375 level in cells. Further, reduced immunopositive cells of Ki-67, p-PI3KCA ^Tyr317, and p-AKT ^Ser473 were observed by the treatments of FN, while the intracellular Bax- and Apaf-1-positive cells were increased dose-dependently. Beneficially, FN-treated tumor-bearing mice exhibited reduced tumor mass and intercellular miR-375 expression. Meanwhile, immuno-labeled cells and proteins of Bax, Caspase-3, and Apaf-1 in FN-treated mice were increased dose-dependently, whereas ERα, p-PI3KCA ^Tyr317, and p-AKT ^Ser473 positive cells and proteins were downregulated, respectively. Collectively, our current results elucidate that FN exerts effective therapeutic benefits against OGS, and the pharmacological mechanism may be related to promoting cell apoptosis by inactivating intracellular miR-375/ERα−PI3K/AKT cascaded pathway.     

In vitro and in vivo studies of RNAse of Bacillus intermedius

Cytotoxicity of RNAase from Bacillus intermedius was studied in vitro and in vivo. It was shown that the enzyme had slightly pronounced cytotoxicity according to the tests with inhibition of cell proliferation and biosynthesis of cell nucleic acids. The RNAase was also shown to impair the vital staining by neutral red. The efficiency of the impairment much more depended on the enzyme catalytic activity than on the proliferation and biosynthesis of nucleic acids. In vivo toxicity of RNAase from B. intermedius was 3-5 times higher than that of pancreatic RNAase. Possible mechanisms of the different toxicity of the enzymes are discussed.