The preparation and use of pyridoxal [32P]phosphate as a labeling reagent for proteins on the outer surface of membranes.

Pyridoxal [32P] phosphate was prepared using [gamma-32P] ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [32P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [32P] phosphate. The Schiff base formed between pyridoxal [32P] phosphate and protein amino groups was reduced with NaBH4. The distribution of pyridoxal [32P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

Synthesis of low molecular weight mimetics of heparin

Natural hexosaminoglycan heparin remains the most commonly prescribed anticoagulant in hospitalized patients. However its administration could induce side clinical events, including thrombocytopenia and bleeding. This explaines the need of development of alternative anticoagulant drugs based on modified heparin and polyanionic oligo- and polysaccharide derivatives, such as sulfated glucans, phosphomannans and fucoidans. Here we review the works on the synthesis of oligosaccharides related to low molecular weight hepain fragments and their derivatives, as well as oligosaccharides, which imitate parts of heparin chain responsible for biological activity. These works were aimed to develop the pharmaceutical preparations lacking ofheparin disadvantages.     

Synthesis of [gamma-32P]thiamine triphosphate

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and thiamine diphosphate in the presence of ethyl chloroformate. After purification by two ion-exchange purification steps, the thiamine derivative has a specific radioactivity of 10 Ci/mmol. The average final yield synthesis is about 10%.     

Synthesis,activity and molecular modeling of a new series of chromones as low molecular weight protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTP) are crucial elements in eukaryotic signal transduction. Several reports suggested that the LMW-PTP family has oncogenic relevance. Moreover, LMW-PTP has been recognized as a negative regulator of insulin-mediated mitotic and metabolic signaling. Thus, inhibition of the LMW-PTP can be considered an attractive approach for the design of new therapeutic agents for the treatment of type II diabetes and for new antitumoral drugs. To date very few (and weak) inhibitors of LMW-PTP have been identified. On the basis of the reported weak activity of some flavonoids on phosphatases, we discovered a lead that originated a new class of highly active LMW-PTP inhibitors; these compounds inhibit also PTP-1B and are active in cellular assays. Docking experiments and SAR highlighted the possible binding mode of these compounds to the enzyme, putting the background for the future optimization of their inhibitory activity and selectivity towards the closely related enzyme PTP-1B.     

Dexamethasone-conjugated low molecular weight polyethylenimine as a nucleus-targeting lipopolymer gene carrier

Dexamethasone, a glucocorticoid steroid, can dilate the nuclear pore complexes and translocate into the nucleus when it is bound to its glucocorticoid receptor, suggesting that the transport of DNA into the nucleus may be facilitated by the reagent. In this research, dexamethasone was conjugated to low molecular weight polyethylenimine (2 kDa) for efficient translocation of the polymer/DNA complex into the nucleus. Polyethylenimine (PEI)-dexamethasone (PEI-Dexa) was synthesized by one-step reaction using the Traut's reagent. In gel retardation assay, the PEI-Dexa/DNA complex was completely retarded at or above 0.3/1 weight ratio (polymer/DNA). The average size distributions and zeta-potential values of the complexes were measured at various weight ratios. In vitro transfection assay showed that the PEI-Dexa/DNA complex had higher gene delivery efficiency compared to PEI 2kDa/DNA complex. The localization of PEI-Dexa/plasmid DNA complexes in the nucleus was confirmed by using total internal reflection fluorescence and Nomarski differential interference contrast microscope as well as confocal microscope. Therefore, with efficient nuclear translocation and low cytotoxicity, PEI-Dexa may be useful for nonviral gene therapy.     

Isolation of a 32P-labeled polypeptide of low molecular weight from phosphorylated human erythrocyte membranes.

The incorporation of 32P into well washed human erythrocyte membranes was studied in a medium containing [gamma-32P]ATP, Mg2+, and EGTA. Following phosphorylation, the membranes were completely solubilized in 1% sodium dodecyl sulfate and subjected to gel electrophoresis in dodecyl sulfate polyacrylamide. A large incorporation of radioactivity was observed in a band which migrated faster than component 7 (nomenclature of T. L. Steck, (1972), J. Mol. Biol. 66, 295) but slower than the bromophenol blue tracking dye, and did not stain with Coomassie Blue. Isolation of this band by preparative gel electrophoresis revealed that 41% of the radioactivity was associated with a 32P-labeled polypeptide. This polypeptide was further purified by gel chromatography on Sephadex LH-20 in chloroform-methanol-HCl, and Bio-Gel A 1.5m in dodecyl sulfate. Its amino acid composition is characterized by a high content of acidic residues. The calculated minimal molecular weight is 15084. Based upon the recovery of amino acids, the polypeptide fraction comprises at least 1.8% by weight of the total erythrocyte membrane proteins. An apparent molecular weight of 15000 was estimated by gel chromatography in dodecyl sulfate, while a range of 14000-16000 was estimated by electrophoresis in dodecyl sulfate polyacrylamide. The state of phosphorylation of this peptide may reflect a physiological function in the intact red cell.     

Synthesis of low molecular weight compounds with complement inhibition activity

An attempt was made to synthesize a series of non-cytotoxic low molecular weight meta-substituted aromatic ethers (2-4, 5-7) and some of their bioisosteres (14-16) and to evaluate their activity on the activation of human complement (classical pathway) and their intrinsic hemolytic activity. The in vitro assay results of the inhibition of complement-mediated hemolysis by these analogues indicate that the aldehydic meta substituted aromatic ethers show inhibitory potency, while carboxylic acid meta substituted aromatic ethers show hemolytic activity. Some of the bioisosteres exhibit both inhibitory as well as hemolytic property.     

Synthesis of [(99m)Tc]DTPA-folate and its evaluation as a folate-receptor-targeted radiopharmaceutical

A DTPA-folate conjugate was radiolabeled with (99m)Tc by stannous chloride reduction of [(99m)Tc]sodium pertechnetate in an aqueous solution of DTPA-folate. The radiochemical purity of the product consistently exceeded 97%, as assessed by thin-layer chromatography employing conditions analogous to those for radiochemical quality control of the radiopharmaceutical [(99m)Tc]DTPA. HPLC demonstrated that the radiolabeled product resulted from the intact DTPA-folate conjugate and not unconjugated DTPA. The ability of [(99m)Tc]DTPA-folate to target folate receptors in vivo was assessed in biodistribution studies with athymic mice bearing subcutaneous folate-receptor-positive human KB cell tumors. As an internal control, previously studied [(111)In]DTPA-folate was coinjected with the [(99m)Tc]DTPA-folate, along with varying amounts of DTPA-folate (0.38 mg/kg, 1.6 mg/kg, or 14 mg/kg). At each DTPA-folate dose, [(99m)Tc]DTPA-folate exhibited tumor uptake comparable to that of the coadministered [(111)In]DTPA-folate, with radiotracer levels declining at the higher DTPA-folate doses due to competitive receptor binding of the unlabeled conjugate. Tumor uptake of both tracers was also competitively blocked by preadministered folic acid dihydrate (2.9 mg/kg). Tumor-to-background tissue contrast obtained with [(99m)Tc]DTPA-folate was generally similar to that obtained with [(111)In]DTPA-folate. The (99m)Tc-labeled DTPA-folate conjugate may have utility as a targeted radiopharmaceutical for imaging neoplastic tissues known to overexpress the folate receptor.     

低分子量神经丝蛋白（NF—L）的体外组装

Neurofilaments were isolated from bovine spinal cords by ultra-speed centrifugation and examined by negative staining. The neurofilament triplet proteins: NF-L, NF-M and NF-H were purified by DE-52 anion exchange chromatography in the presence of 6 mol/L urea. The reassembly of NF-L under controlled conditions was studied. NF-L can reassemble into 10 nm width filaments within 60 minutes at physiological condition of around 0.15 mol/L NaCl, 2 mmol/L MgCl2, neutral pH(pH 6.8) and 37 degrees C. In 6 mol/L urea, NF-L was examined as 12 nm-diameter particle by low angle rotary shadowing. When dialyzed against reassembly buffer for 20 minutes, some irregular filaments were formed. Further dialyzed for another 40 minutes, the long smooth filaments appeared. Some filaments were unraveled at the end regions, where existed 2-4 subfilaments. Four subfilaments were more often observed. That is to say, the 10 nm-width filament was composed of 4 subfilaments. While dialyzed against the alkaline buffer containing 0.15 mol/L NaCl, NF-L reconstituted into 45-180 nm-long, 10 nm-width filaments, which were not able to elongate into long filaments.     

32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor

A 32P-labelled ATP analog, 3'-O-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P2Y-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831-835), has been used as a probe for the P2Y-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (KD approximately 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate greater than adenosine 5'-O-(2-thiodiphosphate) greater than BzATP greater than ATP greater than beta,gamma-methyleneadenosine 5'-triphosphate greater than 5'-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a P2Y-purinergic receptor. Guanine nucleotides (guanosine 5'-O-(3-thiotriphosphate) greater than GTP greater than guanosine 5'-O-(2-thiodiphosphate) greater than GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32P] BzATP-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 53,000 Da. Photolabeling was inhibited in a concentration-dependent manner by ATP and ADP analogs with a potency order characteristic for a P2Y-purinergic receptor and was modulated by guanine nucleotides. A protein of approximately 53,000 daltons was also labeled by [32P]BzATP in membranes from several other tissues known to express the P2Y-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P2Y-purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the P2Y-purinergic receptor.     

Quantification of absolute Ras-GDP/GTP levels by HPLC separation of Ras-bound [(32)P]-labelled nucleotides

Ras guanine nucleotide binding protein (GTPase) activation is a widely assessed readout in cell biological studies. We describe an improved approach for the quantitative analysis of total GDP and GTP bound to Ras. The present method involves HPLC separation and online detection/quantitation of Ras-bound [(32)P]-labelled GDP/GTP. As compared to standard approaches that are time consuming and/or provide only semi-quantitative data, this technique allows the rapid processing of large numbers of samples for the quantitative determination of Ras-bound GDP and GTP.     

Synthesis and in-vitro evaluation of novel low molecular weight thiocarbamates as inhibitors of human leukocyte elastase

A series of novel low molecular weight thiocarbamate esters (1e-6e) were synthesized and evaluated as inhibitors of human leukocyte elastase (HLE). The thiocarbamate esters studied consist of a substituted primary or secondary aliphatic or aromatic amine and a 1-phenyl-1H-tetrazole-5-thiol (Table I). The HLE catalyzed hydrolysis of N-methoxysuccinyl- L-Ala-L-Ala-L-Pro-L-Val-p-nitroanilide substrate was utilized as the measure of inhibition. N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (1e) exhibited the highest inhibitory activity (k(obs) /[I] = 2.1 x 10(5) M(-1). min(-1) ) and N-allyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (2e) (K(obs) /[I] = 6.1 x 10(4) M(-1). min(-1) ) exhibited the second highest inhibitory activity of all the thiocarbamates. The aromatic N-phenyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (4e) showed the lowest inhibitory activity (K(obs) /[I] = 1.9 x 10(2) M(-1). min(-1) ) among the N-monosubstituted derivatives, similar to that of N-ethyl-N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (5e) (K(obs) /[I] = 1.8 x 10(2) M(-1).min(-1) ). The N-isopropyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (3e) (K(obs) /[I] = 3.3 x 10(3) M(-1).min(-1) ) was about 10 fold more active than (4e) and N, N-diisopropyl, 1-phenyl-1H-tetrazole- 5-thiocarbamate (6e) showed no inhibitory activity against HLE. In the present work less than 3% of HLE specific activity was regained after 24 hours incubation with each of the tested N-monosubstituted thiocarbamates (1e-4e). The time-dependent inhibition of HLE by the thiocarbamate compounds (1e-5e) seems to involve the interaction and possible chemical modification of one enzyme residue. Straight chain nonpolar aliphatic substituents on the nitrogen of the thiocarbamate functionality may be essential for high inhibitory activity. As the degree of substitution (branching) on the nitrogen of the thiocarbamate functionality increases the inhibitory activity of the compounds decreases. The time-dependent inhibition of HLE and the slow deacylation rates by the N-monosubstituted thiocarbamates are consistent with irreversible inhibition.     

Kinetics of low molecular weight substrate hydrolysis by immobilized trypsin]

The catalytic properties of trypsin immoblized on silochrome were studied in a flow reactor with replacement. The hydrolysis of methyl ester N-n-tosyl-L-arginine obeys the Michaelis--Menten kinetics. The apparent K'm value for the system with immobilized trypsin is considerably lower than for native trypsin. The K'm value was decreased with an increase in the rate of the substrate flow through the reactor or when smaller-sized silochrome granules were used. It is assumed that the apparent K'm value for the immobilized system is due to diffusion. The effects of diffusion on the catalytic properties of the immobilized enzyme were estimated.     

The preparation and use of pyridoxal [32P] phosphate as a labeling reagent for proteins on the outer surface of membranes

Pyridoxal [³²P] phosphate was prepared using [γ-³²P]ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [³²P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [³²P] phosphate. The Schiff base formed between pyridoxal [³²P] phosphate and protein amino groups was reduced with NaBH₄. The distribution of pyridoxal [³²P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

Synthesis of the low molecular weight heat shock proteins in plants

Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation of these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticum aestivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots.     

A requirement for albumin as carrier for low molecular weight leukocyte chemotactic factors

Using a micropore filter assay, low molecular weight leukocyte chemotactic factors such as fatty acids, formyl-methionyl peptides, and peptides purified from dextran-activated plasma attract leukocytes suspended in albumin-containing media, but not cells suspended in media lacking in protein or containing non-albumin proteins. These factors become bound to albumin and can be eluted with it from Sephadex columns. The albumin-chemotactic factor complex attracts leukocytes more strongly than either the albumin or the chemotactic factor alone. It is suggested that albumin presents the chemotactic factors at the cell membrane in a form suitable to initiate a response which is not achieved by the same factors in free, low molecular weight form.     

Synthesis of low molecular weight RNA components in cells with a temperature-sensitive polymerase II

AF 8 cells are a mutant cell line of baby hamster kidney cells with a temperature-sensitive polymerase II activity. When these cells grow at the non-permissive temperature (40 degrees C) the syntheseis of low molecular weight RNA components D, C and A is preferentially inhibited, whereas the synthesis of rRNA, tRNA, 5 S RNA and component L is affected only a little or not at all. These results indicate that polymerase II catalyzes the synthesis of components D, C and A.     

The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP.

A method has been developed for the enzymatic preparation of alpha-(32)P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [(32)P]AMP, and cyclic [(32)P]GMP of high specific radioactivity and in high yield from (32)Pi. The method also enables the preparation of [gamma-(32)P]ATP, [gamma-(32)P]GTP, [gamma-(32)P]ITP, and [gamma-(32)P]-dATP of very high specific activity and in high yield. The preparation of the various [alpha-(32)P]nucleoside triphosphates relies on the phosphorylation of the respective 3'-nucleoside monophosphates with [gamma-(32)P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5'-(32)P]nucleoside monophosphates. The [5'-(32)P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [alpha-(32)P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic [(32)P]AMP and cyclic [(32)P]GMP are also prepared enzymatically from [alpha-(32)P]ATP or [alpha-(32)P]GTP by partially purified preparations of adenylate or guanylate cyclases. With the exception of the cyclases, all enzymes used are commerically available. The specific activity of (32)P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [alpha-(32)P]ATP and from 5800 to 6500 Ci/mmol for [gamma-(32)P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [alpha-(32)P]nucleoside triphosphates. Methods for the use of the [alpha-(32)P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other (32)P-labeled compounds of biological interest. Moreover, the [alpha-(32)P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [gamma-(32)P]nucleoside triphosphates should be useful in the study of phosphate transfer systems.     

The use of anti-AHP as a test reagent on blood group documentation cards]

In principle it is possible to use anti-AHP upon bedside test cards for the purpose of blood group documentation. In accordance with the individual quality of the anti-AHP substance an optimal concentration has to be determined and retained. With regard to the rapidity of responses and their strength the anti-AHP involves advantages towards the human anti-A test serum. However, the reliability of reading the results will be slightly limited by the appearance of very fine pseudoagglutinates towards O and B blood corpuscles.     

Synthesis of 6 alpha-tritiated beta-muricholic acid for use as a molecular probe.

Regioselective oxidation of methyl beta-muricholate to give the 6-ketoderivative is described. Stereoselective reduction of this ketone with tritiated NaBH4 furnishes labeled methyl beta-muricholate. The structure of all compounds was confirmed by infrared, 1H, and 13C nuclear magnetic resonance spectroscopy. Data obtained by circular dichroism and mass spectroscopy were in agreement with the structure of the ketone 3.