Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids,polyamines, and structurally related compounds

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mm β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ?-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.     

Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Racemization of the aqua-{N-salicylidene-(S)-alaninato}copper(II) by reaction with potassium cyanate

Copper(II) N-salicylidene-(S)-alaninate trihydrate reacting as the S-enantiomeric parent compound with KOCN in hot diluted methanol yielded by slow crystallisation from the cooled reaction mixture (in the course of 1 day) the racemic product K[Cu{sal-(RS)-ala}(NCO)]. The parameters of the axial type EPR spectrum in X-band region and the LF band position in the electronic spectrum are typical of an axially distorted square pyramidal coordination of the Cu(II) atom in this complex. The spectral properties of the complex cuprate prepared and its basal crystallographic data are consistent with those of the earlier studied¹⁵ K₂[Cu₂{sal-(RS)-ala}₂(μ-NCO)₂] synthetized by using [Cu{sal-(RS)-ala}(H₂O)].H₂O as the racemic parent complex in the reaction mixture with KOCN.     

Histidine utilization by Alcaligenes eutrophus: Regulation of histidase formation under heterotrophic conditions of growth

Histidine supported good growth of Alcaligenes eutrophus strain H 16 as a nitrogen source, but only poor growth as a carbon and energy source. The facultative chemolithoautotrophic bacterium was also able to utilize urocanic acid, the first intermediate of histidine catabolism. The products of histidine degradation were ammonium, formate and glutamate. Three enzymes of the pathway, histidase, urocanase and formiminoglutamate hydrolase, were present in histidine-grown cells. Two types of spontaneous mutants, derived from the wild type, were characterized by an increased growth rate on histidine. One of these types was found to produce histidase constitutively and at a higher activity compared with the parental strain. The second type of mutant had apparently gained an improved histidine uptake system, which is supposed to be growth rate-limiting in the wild type. From the physiological studies the conclusion was drawn that the control of histidine-degrading enzymes is based on induction by urocanate and catabolite repression by carbon sources supporting fast growth, such as succinate or pyruvate. Ammonium was found not to affect catabolite repression, however, we obtained evidence that histidine uptake is subject to a nitrogen control.Abbreviation CTAB hexadecyltrimethylammonium bromide     

Isethionate and taurine transport sites at brain cell membranes: Influx studies with brain slices

The influx of [¹⁴C]isethionate (ISA) into rat brain slices was studied with and without taurine. This influx was relatively rapid, but took place largely by a non-saturable, passive mechanism, which transferred much less ISA into the brain cells than taurine. Taurine inhibited the influx of ISA competitively (K _m=50 and 100 mol/l) at low ISA concentrations, and ISA that of taurine non-competitively (V=200 and 400–700 mol×min^–1×kg^–1 wet weight) at high taurine concentrations. It thus appears that ISA and taurine may have a small number of common transport sites at brain cell membranes, but these are apparently of little significance for the total transport of ISA.     

Comparison of Four Enrichment Media in the Recovery of Campyloracter Jejuni

The effectiveness of 4 enrichment media for the recovery of low levels of inoculated cells of Campylobacter jejuni was evaluated. The media contained antibiotics or antibiotics and bile acids as selective compounds. Three of the media recovered most of the inoculated low numbers of 6 C. jejuni strains. In the 3 media the growth rate of 3 strains, indicated by the increase in the log number of cells during 24 h or 48 h incubation at 42 ° C, was about the same as in the control medium without selective compounds. The same 3 media also recovered a low number of Campylobacter cells from artificially contaminated raw milk or ground meat samples. The enrichment medium B containing 40 I.U. Colistin, 5 μg novobiocin, 2 mg Na-cholic acid and 50 mg cycloheximide per ml was inhibitory for most Campylobacter strains studied.     

Retrograde Inhibition of Transmitter Release by ATP

Abstract: After labelling ACh tissue stores in Torpedo electric organ prisms with radioactive acetate, the release of ACh and ATP triggered by electrical stimulation or KCI depolarization was measured in the same perfusate samples. The luciferin-luciferase reaction for ATP was first counted, then the radioactive content of the sample determined. Further evidence showing that ATP release resulted from postsynaptic transmitter action was that carbachol could induce the release of ATP. A dose-response curve was obtained. Curare or α-bungarotoxin block the release of ATP elicited by carbachol. When triggered by KCI depolarization the increased efflux of ACh and ATP returned to low levels in spite of the maintained depolarization. After two successive KCI depolarizations, it was possible to dissociate the release of both substances. The efflux of ATP was exhausted while ACh release was maintained. If the second KCI depolarization was delayed ATP release recovered, but the release kinetics of ACh and ATP were sustained. The exhaustion of endogenous ATP release or the action of exogenous ATP had little or no effect on the release of ACh triggered by KCI depolarization. On the contrary, the release of ACh induced by electrical stimulation was sensitive to the action of adenine nucleotides, and a quantitative estimation of the inhibition of ACh release by ATP and adenosine could be made. At the onset of stimulation ATP release predominated, being gradually replaced by adenosine, which can be reuptaken. This would terminate the inhibitory action of the nucleotide. Carbachol inhibits evoked ACh release, while the effect of α-bungarotoxin was to increase spontaneous ACh release. These effects could be respectively mediated by an increased or a reduced release of ATP resulting from the postsynaptic action of ACh agonists or antagonists. However, a direct presynaptic effect of these substances is not excluded. It seems possible that the action of ATP on ACh release can be explained through its inhibition of the depolarization-evoked Ca²⁺ entry.     

Synaptosomal uptake

Using purified synaptosomal preparations from rat brain, the uptake ofl-tryptophan and norepinephrine was studied. We were unable to replicate some of the results of the experiments obtained with crude mitochondrial, fractions (P₂). Thus we examined the validity of the results of uptake studies obtained with the crude synaptosomes and established conditions which would simulate the biochemical milieu in which the nerve terminals functionin vivo, such as active substrate-dependent respiration, respiratory coupling on addition of ADP, low impurity of noncharacteristic markers, exogenous added proteins (e.g. bovine serum albumin), and verification by electron microscopy. All uptake studies withl-TRP and NE were completed in a system designed for simultaneous recording of respiration and the effect of added ADP. This system was also employed in comparative studies with mitochondria purified by multiple density gradients derived either from the perikaryon or from synaptosomes. Synaptosomal or mitochondrial preparations which did not conform to the above criteria invariably showed significantly lowered ability of uptake ofl-TRP or NE. This was found to be related to impairment in their respiratory and coupling ability. When the experimental conditions of others were employed, the time course of uptake of TRP for crude synaptosomes (P₂) was 100 nmol/g/min and was linear for 2.5 min, while for the purified synaptosomes it was 20 nmol/g/min with a l-min linearity. The mitochondria purified from P₂ displayed 30 nmol/g/min uptake withl-TRP with a linearity of 2.5 min. Reconstituted system of purified synaptosomes and mitochondria gave 60 nmol/g/min ofl-TRP transport with 2.5 min linearity. Also examined was the effect of eight different media. It was found that Krebs-Ringer solution containing glucose (40 mM), pyruvate and malate (10 mM), and ADP (250 nmol) gave optimal uptake of TRP both for synaptosomes and for mitochondria, increasing it to 60 and 86 nmol/g/min. The above conditions also enhanced the uptake of NE by synaptosomes and mitochondria. Uptake of NE was not proportional to protein concentration when the protein content exceeded 0.4 mg. Purified synaptosomal mitochondria accumulated NE more actively than the purified nonsynaptic free mitochondria, albeit at the same rate. Synaptic and free mitochondria had an impaired uptake of NE in presence of DNP, antimycin A, and rotenone, and unlike withl-TRP, pyruvate and malate also reduced uptake of NE. Significant differences were noted for the cytochrome oxidase activity between the synaptosomal and free michondria when compared to that of the homogenate.