Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced “spontaneous” DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H₂O₂-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine–protein kinase ABL1 (c-ABL) and Heat shock 70 kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABL^T315I). We also proved that Apg-2 inhibition aggravated H₂O₂ induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABL^T315I to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development.     

Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr–Abl oncoprotein fused to the cytoplasmic transduction peptide

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to localize in the cytoplasmic compartment and is, therefore, applicable for cytoplasmic targeting. The Bcr–Abl fusion protein, playing major causative role in chronic myeloid leukemia (CML), is a cytoplasmic oncoprotein that contains an N-terminus oligomerization domain (OD) mediating homodimerization of Bcr–Abl proteins, and an intact OD in Bcr–Abl is required both for the activation of its transforming activity and tyrosine kinase. Therefore, disrupting Bcr–Abl oligomerization represents a potential therapeutic strategy for inhibiting Bcr–Abl oncogenicity. In this study, we explored the possible homodimerization-disrupting and tyrosine kinase inhibiting effect of the transduction of OD in Bcr–Abl positive K562 cells. By expressing in Escherichia coli a CTP-OD-HA fusion protein followed by Ni⁺–NTA affinity purification, immunoblot identification and enterokinase cleavage, we showed that the CTP-OD-HA protein was structurally and functionally active in that it potently transduced and primarily localized into the cytoplasmic compartment, heterodimerized with Bcr–Abl, and potently inhibited the phospho-tyrosine pathways of Bcr–Abl oncoprotein at a low concentration of 4 μM. These results delineate strategies for the expression and purification of therapeutic molecules for intracytoplasmic protein based therapeutics and the CTP-OD-HA-mediated killing strategy could be explored as a promising anti-leukemia agent or an adjuvant to the conventional therapeutic modalities in chronic myeloid leukemia, such as in vitro purging.     

An optimized protocol for detection of <Emphasis Type="Italic">E. coli</Emphasis> β-galactosidase in lung tissue following gene transfer

Staining by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) typically detects activity of E. coli β-galactosidase (β-gal) in transduced tissues that express the LacZ reporter gene. In lung tissue from mice that received β-galactosidase-expressing adeno-associated virus (AAV) vectors via intranasal inhalation, we observed only a low frequency of positive cells after X-gal staining in contrast to other reporter genes, such as alkaline phosphatase or green fluorescent protein. In this study, we systematically tested a number of parameters to improve the sensitivity of X-gal staining in lungs transduced with β-galactosidase-expressing AAV2/5 vectors. We observed that the use of nuclear-targeted LacZ instead of cytoplasmic LacZ as the reporter gene substantially increases the number of positive cells after X-gal staining. The pH of the staining solution determines staining sensitivity and background staining with pH 7.0 resulting in high sensitivity and no background levels. Glutaraldehyde at 0.2% or 0.5% in PBS as fixative provides optimal results for X-gal staining. The alternative substrate, Bluo-gal, showed no improvement compared with X-gal but instead caused nonspecific background staining. We further stained intact fixed lungs with X-gal and processed them for paraffin embedding or cryosectioning, resulting in equal staining intensities. However, en bloc staining of intact tissues resulted in the absence of positive cells within deeper-located lung areas.     

Gemini,a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression

Background

The development of collections of quantitatively characterized standard biological parts should facilitate the engineering of increasingly complex and novel biological systems. The existing enzymatic and fluorescent reporters that are used to characterize biological part functions exhibit strengths and limitations. Combining both enzymatic and fluorescence activities within a single reporter protein would provide a useful tool for biological part characterization.

Methodology/Principal Findings

Here, we describe the construction and quantitative characterization of Gemini, a fusion between the β-galactosidase (β-gal) α-fragment and the N-terminus of full-length green fluorescent protein (GFP). We show that Gemini exhibits functional β-gal activity, which we assay with plates and fluorometry, and functional GFP activity, which we assay with fluorometry and microscopy. We show that the protein fusion increases the sensitivity of β-gal activity and decreases the sensitivity of GFP.

Conclusions/Significance

Gemini is therefore a bifunctional reporter with a wider dynamic range than the β-gal α-fragment or GFP alone. Gemini enables the characterization of gene expression, screening assays via enzymatic activity, and quantitative single-cell microscopy or FACS via fluorescence activity. The analytical flexibility afforded by Gemini will likely increase the efficiency of research, particularly for screening and characterization of libraries of standard biological parts.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis was expressed as Hsp70 fusion protein from the construct pPIC9K/hsp70-TLE in the yeast Pichia pastoris. By fusing gloshedobin to the C-terminus of Hsp70, an expression level of 44.5 mg Hsp70-gloshedobin per liter of culture was achieved by methanol induction. The fusion protein secreted in the culture medium was conveniently purified by two chromatographic steps: Q-Sepharose FF and Superdex 200. The purified enzyme had an apparent molecular mass of 98 kDa according to SDS–PAGE analysis, and exhibited fibrinogenolytic activity that preferentially degraded fibrinogen α-chain. The enzyme also degraded fibrinogen β-chain to a lesser extent, while showing no degradation toward the γ-chain. A fibrinogen clotting activity of 499.8 U/mg was achieved by the enzyme, which is within the range reported for other thrombin-like enzymes. Hsp70-gloshedobin had strong esterase activity toward the chromogenic substrate Nα-p-tosyl-Gly-Pro-Arg-p-nitroanilide, and this activity was optimal at pH 7.5 and 50 °C, and was completely inhibited by PMSF, but not by EDTA. We concluded that Hsp70 has no effect on the physiochemical and biochemical properties of gloshedobin. Although applying a fusion partner with very big molecular weight is unusual, Hsp70 proved its advantage in soluble expression of gloshedobin without affecting its fibrinogenolytic activity. And this positive result may provide an alternative strategy for the expression of thrombin-like enzymes in microbial system.     

Differential in vitro metabolism of β-endorphin in schizophrenia

Biologically active peptide fragments derived from the proteolytic cleavage of β-endorphin (βE) have been shown to be present in the brain. Based on clinical results using some of these fragments in neuropsychiatric disease studies we investigated the in vitro metabolism of βE by twice-washed membrane homogenates of postmortem putamen from sex and age matched controls versus subjects with a diagnosis of schizophrenia. The present study demonstrates that frozen (−80°C) postmortem human tissues are viable for these studies and that metabolism in control tissue proceeds similarly to fresh tissues. Furthermore, a significant increase in the formation of the putative neuroleptic-like peptide fragment desenkephalin-γ-endorphin in postmortem schizophrenic putamen versus controls was shown. A significant decrease in the formation of βE 6–21 was also reported. These data suggest that an approach using postmortem human brain is possible in studying β-endorphin catabolism and is therefore applicable to other neuropeptide systems.     

Potential of Ginkgo biloba L. leaves in the management of hyperglycemia and hypertension using in vitro models

Leaves from four different Ginkgo biloba L. trees (1 and 2 – females; 3 and 4 – males), grown at the same conditions, were collected during a period of 5 months (from June to October, 2007). Water and 12% ethanol extracts were analyzed for total phenolics content, antioxidant activity, phenolic profile, and the potential in vitro inhibitory effects on α-amylase, α-glucosidase, and Angiotensin I-Converting Enzyme (ACE) enzymes related to the management of diabetes and hypertension. The results indicated a significant difference among the trees in all functional benefits evaluated in the leaf extracts and also found important seasonal variation related to the same functional parameters. In general, the aqueous extracts had higher total phenolic content than the ethanolic extracts. Also, no correlation was found between total phenolics and antioxidant activity. In relation to the ACE inhibition, only ethanolic extracts had inhibitory activity.     

Characterization of in vitro proteolytic processing of β-endorphin by reversed-phase HPLC

In vitro, central and peripheral proteolytic processing of β-endorphin by membrane-bound enzymes results in the formation of specific active fragments that have been recently shown to function in behavior, intestinal motility and in the central control of urinary bladder activity. A high resolution, reversed phase high performance liquid chromatography system capable of separating 28 β-endorphin related fragments simultaneously was used to study the time-course processing of β-endorphin by membrane associated peptidases in the brain and regions of the small intestine. The hypothesis we tested was that a homeostatic balance between α- and γ-type endorphins exists in these tissues. The results of the study show that the rate and quantity of fragments produced between the mucosa and nerve-muscle regions of the small intestine are significantly different. Metabolic rates, pattern, and the ratio of α/γ-type endorphins in the brain were very similar to the nerve-muscle region of the small intestine. This suggests that β-endorphin processing to active fragments is occurring at the nerves of the small intestine and that a specific and similar balance of α/γ-type endorphin exists in the brain and gastrointestinal system at neutral pH.     

Expression, in Escherichia coli, of a soluble human Tumor Necrosis Factor receptor

We have expressed in Escherichia coli a soluble, truncated form of the human 55 kDa Tumor Necrosis Factor (TNF) receptor. For this purpose a plasmid was constructed which contains the extracellular domain of the 55 kDa TNF receptor fused to the coding sequence of the IgG binding domains of protein A from Staphylococcus aureus. The fusion product (TNFR-PA) obtained in E. coli is a soluble protein which bound human TNFα (huTNFα) with high affinity. In ligand-blotting experiments huTNFα bound to a single 52 kDa protein, a molecular mass corresponding to that expected for the monomeric fusion product. In gel filtration experiments binding activity was recovered from fractions that eluted at a volume corresponding to 140–150 kDa. TNFR-PA neutralized huTNFα in an in vitro cytotoxicity assay.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay.Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays.TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Seasonal chemical composition, in vitro fermentation and in sacco dry matter degradation of four indigenous multipurpose tree species in Nigeria

A study was carried out to estimate the nutritive value of four indigenous multi-purpose tree (MPT) species (Enterolobium cyclocarpum, Treculia africana, Gliricidia sepium and Millettia griffoniana) by the evaluation of their seasonal chemical composition, in vitro fermentation and in sacco dry matter (DM) degradation. The main objective of the study was to assess the potential of these indigenous MPT in supplementing the feed of ruminant animals during the dry season when grasses are scarce and their quality generally fall short of animal requirements. Leaf samples were randomly collected from the trees for estimation of DM, crude protein (CP), ether extract (EE), ash, neutral detergent fibre (NDFom), acid detergent fibre (ADFom), lignin (sa), in vitro fermentation and in sacco DM degradation. Samples were collected three times to represent seasonal variations as follows: November: early dry; February: mid-dry and April: late dry seasons. All samples had high CP (160–199 g/kg DM) and moderate fibre concentrations (NDFom, 380–580 g/kg DM; ADFom, 290–400 g/kg DM and lignin (sa), 75–107 g/kg DM). T. africana recorded the highest (180–199 g/kg DM) (P<0.001) CP content throughout the seasons. The values obtained for the in vitro fermentation characteristics and in sacco DM degradation of these indigenous MPT indicated the presence of potentially degradable nutrients in the MPT. Data from this study showed that E. cyclocarpum, T. africana, G. sepium and M. griffoniana have potentials that could be harnessed as feed supplements for ruminant animal production in Nigeria during the dry season.     

Novel in vitro and in vivo inhibitors of prolyl endopeptidase

Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.     

Lack of iNOS expression and NO formation by mononuclear phagocytes from hamsters upon stimulation in vitro

In previous investigations, we demonstrated that hamster alveolar macrophages failed to induce the iNOS and produce NO upon stimulation in vitro. The subject of this study was to examine whether the lack of the iNOS pathway in hamster mononuclear phagocytes is tissue-specific. We investigated iNOS expression and NO production in isolated hamster (Lak:LVG(SYR)BR) monocytes (MO), pleural macrophages (PLM), and peritoneal macrophages (PM) in comparison to isolated rat (Crl:CD®(SN)BR) cells. NO release was measured as nitrite with the Griess reaction upon stimulation with lipopolysaccharide (LPS) and/or interferon-γ (INF-γ). The expression of the iNOS protein was detected with western blot-technique and iNOS mRNA was analyzed by RT-PCR. As expected, rat MO, PLM, and PM released NO upon stimulation with LPS and/or IFN-γ. However, hamster cells were not able to generate detectable amounts of NO. Expression of the iNOS protein and iNOS mRNA was only detected in rat, but not in hamster MO, PLM, and PM. Our data indicate that NO may not belong to the effector system of hamster mononuclear phagocytes.     

Impact of moisture on in vitro germination of Metarhizium anisopliae and Beauveria bassiana and their activity on Triatoma infestans

The in vitro germination of 11 Metarhizium anisopliae and 11 Beauveria bassiana isolates originating from substrates collected in rural peridomestic areas in Central Brazil where triatomines are common was tested. Conidia completed germination up to 24 h after exposure to water activity of >0.99 a_w in all isolates tested. At lower 0.93 a_w germination was delayed but conidia of most isolates germinated at high rates (>98 %) within 216 h of incubation. Activities of 2 M. anisopliae and 2 B. bassiana isolates with different patterns of germination at 0.93 a_w were tested in Triatoma infestans third instar nymphs. There was no relationship between germination kinetics in vitro at 0.93 a_w and their activity in vivo at 98, 75 and 43 % relative humidity (rh). Isolates with accelerated germination at 0.93 a_w were not more virulent at 75 and 43 % rh compared with isolates with retarded or no germination. Highest mortalities were observed at 98 % rh, and they did not exceed 25 % after 25 d incubation at lower 75 and 43 % rh. Isolates that originated from a region with an extensive annual arid period showed no adaptation to lower humidity in their activity against T. infestans.     

Assessment of a brown midrib (BMR) mutant gene on the nutritive value of sudangrass using in vitro and in vivo techniques

The brown midrib (BMR) gene has been reported to reduce the lignin concentration in plants, which contributed to increased fiber digestion in ruminants. Three studies were completed to compare the digestibility of a BMR mutant of sudangrass (sorghum bicolor subsp. Drummondii) versus a non-BMR (‘Piper’) variety when included in diets fed to sheep (Study 1), to complete a rumen in vitro assessment of sheep and lactating cow diets (Study 2), and to compare digestibility when included in the diet fed to lactating dairy cows (Study 3). Four wether sheep were used in a 2 × 2 Latin square experiment (Study 1) with total fecal collection to determine total tract apparent digestibility of pelleted Piper (P) and BMR (P-BMR) sudangrass hays. Forage pellets consisted of either P-BMR or P hay with added urea to meet the maintenance crude protein (CP) requirement of the sheep. Digestibility of organic matter (OM; P<0.01), dry matter (DM; P<0.01), acid detergent fiber (ADF; P<0.05), and neutral detergent fiber (aNDFom; P<0.07) was higher for P-BMR than P sudangrass. In vitro rumen digestibility of aNDFom using cattle rumen fluid was higher at 24 (P<0.01), 48 (P<0.01) and 72 h (P<0.01) of fermentation for P-BMR versus P (Study 2). Four lactating Holstein dairy cows (251 ± 30 days in milk) and fitted with ruminal and duodenal cannulae were used in a 4 × 4 Latin square experiment. Total mixed rations (TMR) contained 180 g/kg DM shredded sudangrass hay and 180 g/kg sliced alfalfa hay, but the proportion of P to P-BMR sudangrass varied as 100:0, 66:34, 34:66, or 0:100. Yields of milk and milk protein were highest at the 66:34 level (Quadratic: P=0.06 and 0.07, respectively), but composition of milk fat, protein and lactose, as well as DM intake, did not differ (Study 3), probably because forestomach and total tract apparent digestion of aNDFom and OM did not differ due to sudangrass source.     

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca-ATPase

Membrane Ca²⁺-ATPase activity was stimulated in vitro separately by T₄ (10⁻¹⁰ M) and by epinephrine (10⁻⁶ M). In the presence of a fixed concentration of T₄, additions of 10⁻⁸ and 10⁻⁶ M epinephrine reduced the T₄ effect on the enzyme. β-Adrenergic blockade with propranolol (10⁻⁶ M) prevented stimulation by epinephrine of Ca²⁺-ATPase activity, but did not prevent the suppressive action of epinephrine on T₄-stimulable Ca²⁺-ATPase. In contrast α₁-adrenergic blockade with unlabelled prazosin restored the effect of T₄ on Ca²⁺-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulations by T₄ of red cell Ca²⁺-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (K_d, 1.2 × 10⁻⁸ M; B_max, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains α₁-radrenergic receptor sites that are capable of regulating Ca²⁺-ATPase activity.