Expression of HOX homeogenes in human neuroblastoma cell culture lines

Mammalian genes containing a class-I homeobox (HOX genes) are highly expressed in the embryonic nervous system. As a first step towards the molecular analysis of the role these genes play in neural cells, we studied the expression of four human HOX genes in five neuroblastoma (NB) cell lines - SK-N-BE, CHP-134, IMR-32, SK-N-SH and LAN-1 - during the process of differentiation induced by treatment with retinoic acid (RA). The four genes, HOX1D, 2F, 3E and 4B, located at corresponding positions in the four HOX loci, share a high degree of sequence similarity with the Drosophila Deformed homeotic gene and constitute a homology group, group 10. One of these genes, HOX1D, is not expressed in the cells used, whereas the other three are highly expressed in untreated and RA-induced NB cells, even though the expression pattern in the various lines is slightly different for the three genes. Our analysis reveals a complex and specific expression pattern in these lines, paving the way to an identification of different NB-cell populations by means of specific HOX gene expression schemes. On the other hand, in every line studied, morphological maturation toward a neuronal differentiated phenotype appears to be associated with increased HOX gene expression.     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide     

Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina Platensis

A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2. Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts). The arrangement rpsB-spacer-tsf resembles that reported for E. coli. The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E. coli counterparts.     

C-terminal peptide identification by fast atom bombardment mass spectrometry.

A previously described technique [Rose, Simona, Offord, Prior, Otto & Thatcher (1983) Biochem. J. 215, 273-277] permits the identification of the C-terminal peptide of a protein as the only peptide that does not incorporate any 18O upon partial enzymic hydrolysis in 18O-labelled water. Formation of chemical derivatives followed by combined g.l.c.-m.s. was used in this earlier work. We now describe the isolation from protein digests, by reversed-phase h.p.l.c., of labelled and unlabelled polypeptides and their direct analysis by fast atom bombardment mass spectrometry. Under the conditions used, the 18O label is retained throughout the separation and analysis, thus permitting assignments of C-terminal peptides to be made. Enzyme-catalysed exchange of label into the terminal carboxy group was found to occur in some cases without hydrolysis of a peptide bond. This effect, which may be exploited to prepare labelled peptides, does not prevent application of the method (two separate digests must then be used). We have applied our method to the analysis of enzymic partial hydrolysates of glucagon, insulin and of several proteins produced by expression of recombinant DNA.     

A study of the hydration and thermodynamics of warm-water and cold-water fish collagens.

The hydrated volumes, Vh, of collagens extracted from various fish species were calculated by using the Simha-Einstein equation, and it was found that the hydration of warm-water fish collagen is greater than that of cold-water fish collagen (halibut). Although the intrinsic viscosities of warm-water fish (bigeye-tuna, carp and catfish) collagens are almost the same, the hydrated volume of bigeye-tuna collagen is approx. 1.5 and 3 times those of carp and catfish collagens respectively. The extent of hydration at 20 degrees C is in the following order: bigeye tuna greater than carp greater than catfish greater than halibut. The various thermodynamic activation parameters (delta G*, delta H* and delta S*) were calculated and it was found that they are useful for determining the exact denaturation temperature. It was calculated that the denaturation temperatures of halibut, bigeye-tuna, carp and catfish collagens are 17, 31, 32 and 26-30 degrees C respectively. The variations of hydration, intrinsic viscosity, denaturation temperature and the thermodynamic parameters with the variation of concentration of catfish collagen were also thoroughly examined. The change of thermodynamic parameters from coiled-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results.     

Insulin proteinase liberates from glucagon a fragment known to have enhanced activity against Ca2+ + Mg2+-dependent ATPase.

We find, contrary to previous reports, that substantial cleavage of glucagon by insulin proteinase occurs at only one region, namely the double-basic sequence -Arg17-Arg18-. Cleavage takes place almost exclusively between these two residues, liberating fragments glucagon-(1-17) and glucagon-(18-29). Others have shown that the fragment glucagon-(19-29) is 1000-fold more efficient compared with intact glucagon, at inhibiting the Ca2+-activated and Mg2+-dependent ATPase activity and the Ca2+ pump of liver plasma membranes. We show that this fragment is not liberated in detectable quantities by our insulin proteinase preparation. On the other hand, others have shown that glucagon-(18-29), though less active than glucagon-(19-29), was still 100-fold more active than glucagon itself in the above-mentioned system. Our observations represent the first demonstration of the release by insulin proteinase of a hormone fragment having enhanced activity, although it has yet to be shown that the activity of this fragment is important in vivo. Since the formation of glucagon-(19-29) from glucagon-(18-29) would involve merely removal of Arg18, a second enzyme might exist to provide the more active fragment.     

Enzyme-assisted semisynthesis of polypeptide active esters and their use.

A method is described for the preparation of polypeptides activated uniquely at the C-terminus. The polypeptide is incubated in a concentrated solution of an amino acid active ester, the latter having its amino group free but adequately protected by protonation. The amino acid ester is coupled via its amino group to the C-terminus of the polypeptide by enzymic catalysis (reverse proteolysis). The resulting polypeptide C-terminal active ester is then isolated and coupled to a suitable amino component (generally a polypeptide) in a subsequent chemical coupling. The method appears to be generally applicable; fragments of horse heart cytochrome c, and porcine insulin, are used as examples. Two new analogues of cytochrome c have been prepared by using this method, with yields of up to 60% in the final coupling. Scope and limitations of the method are discussed.     

Hypothesis: A nicotine-dopamine interaction linking smoking with Parkinson's disease and tardive dyskinesia

1. Nicotine, an important pharmacological component of cigarette smoke, is known to have significant effects on central nervous system (CNS) dopaminergic function. Although acute doses of nicotine have been shown to facilitate dopamine release, recent data indicate that chronic nicotine treatment may actually decrease CNS dopamine turnover in the striatum. 2. A number of epidemiological investigations have demonstrated that individuals who are or who have been smokers are less likely to develop idiopathic Parkinson's disease (a disorder involving a deficit in nigrostriatal dopaminergic neurotransmission). In addition, there is preliminary evidence that individuals with tardive dyskinesia (a hyperkinetic movement disorder observed in some cases of chronic neuroleptic treatment and thought by some to be associated with striatal dopamine receptor supersensitivity) are more likely to be smokers. 3. A unitary hypothesis is presented, proposing that smoking in early adult life may decrease CNS catecholamine turnover, thereby protecting against free radical formation from catecholamine oxidation that in turn damages striatal neurons. These individuals are thereby "protected" from the later development of Parkinson's disease. In this hypothetical scheme, individuals who are given neuroleptics and who also are smokers may develop a greater degree of dopamine receptor supersensitivity due to combined receptor blockade by neuroleptics and a decrease in CNS dopamine turnover caused by nicotine, resulting in an increased prevalence of tardive dyskinesia in this group.     

Sequence identity between an inverted repeat family of transposable elements in Drosophila and Caenorhabditis.

The Tc1-like transposable elements, originally described in Caenorhabditis elegans, have a much wider phylogenetic distribution than previously thought. In this paper, we demonstrate that Tc1 shares sequence identity in its open reading frame and terminal repeats with a new transposable element Barney (also known as TCb1-Transposon Caenorhabditis briggsae 1). Barney was detected and isolated by Tc1 hybridization from the closely related nematode species, Caenorhabditis briggsae. The conserved open reading frames of Tc1 and Barney share identity with a structurally similar family of elements named HB found in Drosophila melanogaster, after the introduction of 3 small centrally located deletions in HB1. These reading frames would code for proteins with 30% amino acid identity (42% when conservative changes are included). Tc1, Barney and HB1 contain highly conserved blocks of amino acids which are likely to be in the functional domains of the putative transposase.