Rabbit Reticulocyte Lysate Cell-free System Research Articles

СИНТЕЗ БІЛКІВ ТЕПЛОВОГО ШОКУ У МІДІЙ MYTILUS EDULIS

Досліджено синтез білків теплового шоку in vivo та in vitro у мідій Mytilus edulis за впливу бурового розчину лігносульфонатного типу. Встановлено, що стресовий білок з молекулярною масою 68 кД можна використовувати як біомаркер забруднення морської води буровими розчинами лігносульфонатного типу у наднизьких концентраціях.

TYchi, a novel chitinase with RNA N-glycosidase and anti-tumor activities

Chitinases which catalyze hydrolysis of chitin are believed to be antifungal proteins in plant. Nevertheless, a variety of functions and some new enzymatic activities of chitinases have been found in recent years. We cloned a novel protein from Trichosanthes kirilowii Maximowicz (Family Cucurbitaceae) named TYchi. Expression of TYchi gene in T. kirilowii plants was induced by F. oxysporum, an important cucurbitaceous fungal pathogen, which indicated that TYchi involved in the pathogen-induced plant defense reaction. In addition to its chitin-hydrolytic activity, the recombinant TYchi protein also had RNA N-glycosidase property. In cell-free rabbit reticulocyte lysate system, TYchi inhibited protein synthesis with an IC50 of approximate 5 nM. TYchi also exhibited efficient cytotoxicities to leukemia U937 and choriocarcinoma JAR cells with IC50 about 54 microg ml(-1) and 73 microg ml(-1), respectively. Structure analyses indicated that the putative domain of TYchi is highly similar to the well known active domain of the N-glycosidase trichosanthin (TCS). This bifuntional protein should be useful in diverse applications like RIP-based immunotoxin agent and genetic engineering of plant resistance.

Cochinin B, a Novel Ribosome-Inactivating Protein from the Seeds of Momordica cochinchinensis

Cochinin B, a novel ribosome-inactivating protein (RIP) with a molecular weight of 28 kDa, was purified from the seeds of Momordica cochinchinensis (Cucurbitaceae). The isolation procedure entailed ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose column and size-exclusion chromatography on Superdex 75 column with a fast protein liquid chromatography (FPLC) system. The first twenty N-terminal amino acid residues of Cochinin B showed homology to type I RIPs from other Momordica species. The purified Cochinin B displayed a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate system with IC50 of 0.36 nM. Furthermore, it exhibited N-glycosidase activity and cytotoxicity against Vero cell line with IC50 higher than 1540 nM. Interestingly, Cochinin B manifested strong anti-tumor activities on human cervical epithelial carcinoma (HeLa), human embryonic kidney (HEK293) and human small cell lung cancer (NCI-H187) cell lines with IC50 of 16.9, 114 and 574 nM, respectively.

A mitogenic defensin from white cloud beans ( Phaseolus vulgaris)

A peptide, with a molecular mass of 7458 Da, was purified from the seeds of white cloud beans ( Phaseolus vulgaris cv. ‘white cloud bean’). This peptide was isolated using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. The peptide had both antifungal and antibacterial activities. It reduced the activity of HIV-1 reverse transcriptase and it also inhibited translation in a cell-free rabbit reticulocyte lysate system. Its antifungal activity was retained after incubation with trypsin but was reduced when the ambient ionic strength was raised. The peptide elicited a mitogenic response from mouse splenocytes but did not stimulate nitric oxide production in mouse macrophages.

Limenin, a defensin-like peptide with multiple exploitable activities from shelf beans

From the seeds of the shelf bean, an antifungal peptide with a molecular mass of 6.5 kDa was isolated. The isolation procedure comprised affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Mono S, and gel filtration on Superdex 75. The peptide was adsorbed on Affi-gel blue gel and Mono S. It potently suppressed mycelial growth in Botrytis cinerea, Fusarium oxysporum, and Mycosphaerella arachidicola with an IC(50) of 2.9, 2.1, and 0.34 microM, respectively. It exerted antibacterial activity toward several bacterial species with an IC(50) approximating 100 microM. [Methyl-(3)H]-thymidine incorporation into isolated mouse splenocytes was stimulated. [Methyl-(3)H]-thymidine incorporation into M1 (myeloma) and L1210 (leukemia) cells was inhibited. The peptide reduced the activity of HIV-1 reverse transcriptase and also inhibited translation in a cell-free rabbit reticulocyte lysate system.

Lunatusin, a trypsin-stable antimicrobial peptide from lima beans ( Phaseolus lunatus L.)

An anti-fungal peptide designated as lunatusin, with a molecular mass around 7 kDa, was purified from the seeds of Chinese lima bean ( Phaseolus lunatus L.). The peptide was isolated using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. Lunatusin exerted an anti-fungal activity toward fungal species such as Fusarium oxysporum, Mycosphaerella arachidicola and Botrytis cinerea, and an antibacterial action on, Bacillus megaterium, Bacillus subtilis, Proteus vulgaris and Mycobacterium phlei. It also inhibited proliferation in the breast cancer cell line MCF-7. Lunatusin reduced the activity of HIV-1 reverse transcriptase and it also inhibited translation in a cell-free rabbit reticulocyte lysate system. Its anti-fungal activity was retained after incubation with trypsin. Lunatusin elicited a mitogenic response from mouse splenocytes.

Vulgarinin, a broad-spectrum antifungal peptide from haricot beans ( Phaseolus vulgaris)

From the seeds of haricot beans ( Phaseolus vulgaris), an antifungal peptide with a molecular mass around 7 kDa was purified by using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. This peptide named vulgarinin manifested an antifungal activity toward fungal species such as Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola and Botrytis cinerea, and an antibacterial action on Mycobacterium phlei, Bacillus megaterium, Bacillus subtilis and Proteus vulgaris. It also inhibited proliferation in leukemia cell lines L1210 and M1 and breast cancer cell line MCF-7. This peptide could reduce the activity of HIV-1 reverse transcriptase and inhibited translation in a cell-free rabbit reticulocyte lysate system. Its antifungal activity was retained after incubation with trypsin.

First report of a glutamine-rich antifungal peptide with immunomodulatory and antiproliferative activities from family Amaryllidaceae

This represents the first report of purification of a glutamine-rich antifungal peptide from family Amarylliaceace. The peptide, designated as nartazin, was purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis by means of ion-exchange chromatography and affinity chromatography. Its molecular mass was 7.1kDa, as determined by SDS–PAGE and gel filtration. Nartazin stimulated proliferation of mouse splenocytes and bone marrow cells but inhibited proliferation of leukemia L1210 cells. It also inhibited translation in a cell-free rabbit reticulocyte lysate system. The sequence of its first 20 N-terminal residues was characterized by an abundance of glutamine. The peptide possessed antifungal activity on four phytopathogenic fungi. Its activity was retained after incubation with bovine trypsin and chymotrypsin (enzyme: substrate ratio 1:10 w/w) at 37°C for 1h but was attenuated after treatment with proteinase K. The data revealed its pronounced resistance to proteolytic digestion.

A polymeric immunoglobulin receptor-like milk protein with inhibitory activity on human immunodeficiency virus type 1 reverse transcriptase

A protein with a molecular mass of 85 kDa and an N-terminal sequence resembling polymeric immunoglobulin receptor has been isolated from bovine milk. The isolation procedure involved removal of globulin from acid whey by precipitation with 1.8 M (NH 4) 2SO 4 followed by addition of (NH 4) 2SO 4 to attain a concentration of 3.6 M. Subsequent steps included chromatography on CM-Sepharose and Mono S and elution of the protein of interest with a linear NaCl concentration gradient. The polymeric immunoglobulin receptor-like milk protein inhibited HIV-1 reverse transcriptase (RT) with an IC 50 of 4.8 μM. However, it did not exhibit ribonuclease activity. Neither did it inhibit translation in a cell-free rabbit reticulocyte lysate system.

Adustin, a small translation-inhibiting polypeptide from fruiting bodies of the wild mushroom Polyporus adusta

A polypeptide, with a molecular mass of 16.5 kDa as determined by gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, has been isolated from the mushroom Polyporus adusta. The polypeptide, designated as adustin, inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC 50 of 0.34 μM. It was isolated using a protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. Adustin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and CM-Sepharose.

Adustin, a small translation-inhibiting polypeptide from fruiting bodies of the wild mushroom Polyporus adusta

A polypeptide, with a molecular mass of 16.5 kDa as determined by gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, has been isolated from the mushroom Polyporus adusta. The polypeptide, designated as adustin, inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 0.34 μM. It was isolated using a protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. Adustin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and CM-Sepharose.

A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju

A 12 kDa ribonuclease preferential for poly U and with much lower activity toward poly A, poly G and poly C was isolated from fresh fruiting bodies of the mushroom Pleurotus sajor-caju. A purification procedure involving ion exchange chromatography on CM-cellulose, affinity chromatography on Red-Sepharose and Heparin-Sepharose, and fast protein liquid chromatography–gel filtration on Superdex 75 was used. The ribonuclease was adsorbed on all of the first three types of chromatographic media. It exhibited some activity toward herring sperm DNA and calf thymus DNA. The ribonuclease activity was unaffected in the presence of KCl (10 and 100 mM) and NaCl (100 mM and 1 M), but was strongly inhibited by CuSO 4 (0.01 and 0.1 mM) and less potently inhibited by other divalent salts including MgCl 2, CaCl 2, ZnCl 2, ZnSO 4 and FeSO 4. The optimal pH was 5.5 and the ribonuclease was stable up to 60 °C for 1 h. The ribonuclease inhibited mycelial growth in the fungi Fusarium oxysporum and Mycosphaerella arachidicola with an IC 50 value of 95 and 72 μM, respectively. Out of the 12 species of bacteria tested, only Pseudomonas aeruginosa and Staphylococcus aureus were inhibited in growth by the ribonuclease. Viability of the tumor cells HepG2 (hepatoma) and L1210 (leukemia) was reduced with an IC 50 of 0.22 and 0.1 μM, respectively in the presence of the ribonuclease. The ribonuclease inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC 50 of 158 nM and 3 H -methyl-thymidine uptake by murine splenocytes with an IC 50 of 65 nM.

Purification of castamollin, a novel antifungal protein from Chinese chestnuts

A novel antifungal protein, designated castamollin, was isolated from Chinese chestnut ( Castanea mollisima) seeds with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM–Sepharose and FPLC-gel filtration on Superdex 75. Castamollin possessed a novel N-terminal sequence demonstrating little similarity to N-terminal sequences of Castanea sativa chitinase. Castamollin exhibited a molecular mass of 37 kDa in gel filtration and SDS–PAGE. It inhibited the activity of human immunodeficiency virus-1 reverse transcriptase with an IC 50 of 7 μM and translation in a cell-free rabbit reticulocyte lysate system with an IC 50 of 2.7 μM. Castamollin displayed antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, Physalospora piricola, and Coprinus comatus but was devoid of lectin activity.

Isolation of cucurmoschin, a novel antifungal peptide abundant in arginine, glutamate and glycine residues from black pumpkin seeds

A novel antifungal peptide, with a molecular mass of 8 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and in gel filtration on Superdex 75 and designated cucurmoschin, was isolated from the seeds of the black pumpkin. The peptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. Cucurmoschin inhibited mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella oxysporum. It inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC 50 of 1.2 μM. The N-terminal sequence of cucurmoschin was rich in arginine, glutamate and glycine residues.

A homodimeric sporamin-type trypsin inhibitor with antiproliferative, HIV reverse transcriptase-inhibitory and antifungal activities from wampee (Clausena lansium) seeds.

A homodimeric trypsin inhibitor with a molecular mass of 54 kDa was isolated from the seeds of Clausena lansium (Lour) Skeels with a very simple procedure comprising extraction with an aqueous buffer and ion exchange chromatography on CM-cellulose. It inhibited trypsin with an IC50 of 2.2 nM but was without any inhibitory effect on chymotrypsin and proteinase K. The uptake of MTT by human leukemia HL60 and hepatoma Hep G2 cells was inhibited with an IC50 of 100 microM. Translation in the cell-free rabbit reticulocyte lysate system was inhibited with an IC50 of 3.6 microM. The activity of HIV-1 reverse transcriptase was reduced in the presence of the trypsin inhibitor. The trypsin inhibitor exerted antifungal activity toward Physalospora piricola but not Mycosphaerella arachidicola, Botrytis cinerea, Fusarium oxysporum or Coprinus comatus.

Contamination of Ribosome Inactivating Proteins with Ribonucleases, Separated by Affinity Chromatography on Red Sepharose

Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity. It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity. It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.

Hispin, a novel ribosome inactivating protein with antifungal activity from hairy melon seeds

A ribosome inactivating protein demonstrating a molecular mass of 21 kDa and a novel N-terminal sequence was isolated from seeds of the hairy melon. The purification procedure involved affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose and Mono S. The protein designated hispin inhibited translation in the cell-free rabbit reticulocyte lysate system with an IC 50 of 165 pM and exhibited N-glycosidase activity. Antifungal activity was also observed.

Isolation of an antifungal thaumatin-like protein from kiwi fruits

A single-chain 21 kDa protein exhibiting antifungal activity against Botrytis cinerea and some suppressive effects on Mycosphaerella arachidicola and Coprinus comatus was isolated from kiwi fruits. The protein, designated kiwi fruit thaumatin-like protein, did not inhibit translation in the cell-free rabbit reticulocyte lysate system but inhibited HIV-1 reverse transcriptase. It was purified to apparent homogeneity using a procedure involving saline extraction, (NH 4) 2SO 4 precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and gel filtration on Superdex 75, respectively.

Solenin, a novel protein with translation-inhibiting activity from the traditional Chinese medicinal fish, the sea dragon Solenognathus hardwickii

A single-chained protein designated solenin was isolated from Solenognathus hardwickii, a fish used as traditional Chinese medicinal material. Solenin was capable of inhibiting translation in a cell-free rabbit reticulocyte lysate system with an IC 50 of 2 μM and expressing a ribonuclease activity of 0.8 U/mg toward yeast transfer RNA, but it lacked N-glycosidase activity characteristic of ribosome inactivating proteins Solenin exhibited a molecular weight of 18 kDa and possessed an N-terminal sequence AHDAEVNEVKAQVAA. The protein was adsorbed on three types of chromatographic media: Affi-gel blue gel, CM-Sepharose and Mono S. It was devoid of antifungal and lectin activities.

Feedback Inhibition of Poly(A)-binding Protein mRNA Translation: A POSSIBLE MECHANISM OF TRANSLATION ARREST BY STALLED 40 S RIBOSOMAL SUBUNITS

An adenine-rich cis element at the 5'-untranslated region (UTR) of Pabp1 mRNA is able to inhibit translation of its own mRNA. Similar inhibition of translation of a reporter beta-galactosidase mRNA is observed when the adenine-rich auto regulatory sequence (ARS) is placed within the 5'-UTR of this mRNA. For this translational control the distance of the ARS from the 5' cap is not important. However, it determines the number of 40 S ribosomal subunits bound to the translationally arrested mRNA. Inhibition of mRNA translation by this regulatory sequence occurs at the step of joining of the 60 S ribosomal subunit to the pre-initiation complex. Translational arrest of the ARS containing mRNA in a rabbit reticulocyte lysate cell-free system in the presence of exogenous Pabp1 protects the 5'-flanking region of the ARS from nuclease digestion. This protection depends on the binding of the 40 S ribosomal subunit to the mRNA. The size and the sequence of the nucleotide-protected fragment depends on the location of the ARS within the 5'-UTR. When the ARS is located at a distance of about 78 nucleotides from the 5' cap, a 40-nucleotide long region adjacent to the ARS is protected. On the other hand, when the ARS is moved further away from the 5' cap to a distance of approximately 267 nucleotides, a 100-nucleotide-long region adjacent to the ARS is protected from nuclease digestion. Nuclease protection is attributed to the presence of one or more stalled 40 S ribosomal subunits near the Pabp1-bound ARS.