Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

The oxidation of cyclothionine by D-aspartate oxidase

Cyclothionine was found to be a substrate for bovine kidney D-Aspartate oxidase. The substrate, prepared chemically as a mixture of the possible stereoisomers, exhibits an inhibition at elevated concentrations. Compounds structurally related to cyclothionine, like TMDA and alpha-alpha'-iminodipropionic acid, have also been assayed with the enzyme.     

Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner.

The activities of the individual hepatitis B virus (HBV) promoters and the effects of the HBV enhancer on these promoters in several human cell types have been compared by measuring the activity and RNA levels of the linked reporter function chloramphenicol acetyltransferase. The relative promoter activities in the human HepG2 (liver), HeLa, and HS27 (fibroblast) cell lines are in the order precore greater than X greater than preS2 greater than preS1; thus, the promoters of the gene producing the largest quantity of viral proteins have relatively low activity. The juxtaposition of the HBV enhancer in either orientation increased the promoter activities only modestly (2- to 5-fold) in the nonliver cell lines, whereas it dramatically increased (20- to 100-fold) the promoter activities in the liver cell line. Thus, the HBV enhancer is especially active in liver cells. This may be one of the causes of hepatotrophicity of the virus.     

Normal rat intestinal cells IEC-18: characterization and transfection with immortalizing oncogenes

IEC-18 cells, a cell line derived from the ileum of rat intestine, have the characteristics of normal cells since they have a contact inhibited cell growth, do not form colonies in soft agar and are not tumorigenic when injected in nude mice. IEC-18 cells were transfected with nuclear oncogenes, c-myc, v-myc and SV40 T antigen in order to obtain immortal cell lines. Independent clones were isolated and characterized for the growth properties. Expression of v-myc altered the morphology of the cells and shortened the doubling time. A slow growth together with a low cloning efficiency was associated with the expression of SV40 T antigen. No changes either in growth or in morphology were observed in c-myc-expressing IEC-18 cells. Expression of these nuclear oncogenes did not result in the neoplastic transformation of the IEC-18 cells, since none of the clones lost the anchorage dependence or were able to form tumors in vivo. The c-myc-containing IEC-18 cells were unable to secrete in the growth medium TGF and exposure to TGF inhibited the growth rate by 30%. All these observations are consistent with the conclusion that the expression of nuclear oncogenes does not lead to the neoplastic transformation of these cells.     

Identification of new products of S-aminoethylcysteine ketimine autoxidation

Summary In continuation of a previous work (Pecci et al., 1993), dedicated to the detection of the autoxidation products of S-aminoethylcysteine ketimine (AECK), we give here data for the identification of 2,3,6,7-tetrahydro-4H-[1,4]thiazino[2,3-b]thiazine, thiomorpholine-3-one and 5,5, 6,6-tetrahydro-2,2-dihydroxy-3,3-bi-2H-thiazine among the products of AECK autoxidation. Identification has been done on the basis of mass spectrometry and NMR spectral analyses of the isolated products.Abbreviations TLC thin layer chromatography - HPLC High performance liquid chromatography - AECK S-aminoethylcysteine ketimine     

Chromatographic determination of arylsulfatases A and B in human gastric mucosa]

In continuation of a previous work, we have confirmed the occurrence of arylsulfatase A in 4 samples of human gastric mucosa analysed by the chromatographic procedure described by Stevens et all. By using the chromatographic method we have also evidentiated the occurrence of arylsulfatase B, which was not detected by using the method of Baum et all. The B form was lower than the A form in 3 samples while it was higher in another sample. In the latter sample of gastric mucosa it was also detected the unusual form Bm of arylsulfatase. It was concluded that both forms A and B of arylsulfatase are present in human gastric mucosa, in variable amounts and that the simple procedure developed by Baum et all., although suitable for the analysis of these enzymes in the urine, is not useful for the determination of arylsulfate B in the gastric mucosa.     

Sulfur-containing cyclic ketimines and imino acids. A novel family of endogenous products in the search for a role.

Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopyruvate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high-affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur-containing cyclic products provided with a possible neurochemical function to be investigated further.     

MK-801 Prevents 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism in Primates

In cynomologus monkeys, systemic administration of MK-801, a noncompetitive antagonist for the N-methyl-D-aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MK-801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK-801, the levels of toxic 1-methyl-4-phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK-801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.     

Dimerization and other changes of aminoethylcysteine ketimine.

High performance liquid chromatography and gas liquid chromatography have been used for the study of the stability of aminoethylcysteine-ketimine (AECK) in different experimental conditions. Concentration and acidic pH lead to the formation of the dimer of AECK which is very sensitive to temperature and is slowly converted to the corresponding decarboxylated dimer even at room temperature. In the presence of air at neutral and alkaline pH AECK is converted to an unknown compound having spectral characteristics similar to AECK and to other compounds not detectable by HPLC. Under nitrogen at neutral pH AECK is more stable and only undergoes the dimerization reaction to some extent.