Antitumour activities of levans produced by Zymomonas mobilis strains

Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

Dependence of ionized and total Ca in squid axons on Nao-free or high- Ko conditions

The level of intracellular Ca in squid axons (both ionized and total Ca) was studied as a function of the experimental variables [Na]i, [Na]o, pHi, cyanide, and depolarization. Ionized Ca was measured by following the light emission of aequorin while total Ca was measured by the atomic absorption analysis of samples of axoplasm. Aequorin glow is known to be increased either by the application of Nao-free solutions or by depolarization produced by external solutions containing greater than normal K concentrations. The present results show that if [Na]i is low, the depolarization that is brought about by solutions with elevated [K] leads to a resting light emission that is decreased rather than increased, as is the case when [Na]i is high. In axons where [Na]i is varied, a comparison of the increments in light emission produced by the application first of Na-free and then of high-K solutions shows that they have an identical dependence on [Na]i, with a half-activation of Ca entry produced by an [Na]i of 25-30 mM. Changes in pHi affect the aequorin signal produced by depolarization, with acidification reducing and alkanization increasing the response. Cyanide did not greatly affect the size of the signal resulting from either Nao removal or that from depolarization.     

Variation in the ribosomal internal transcribed spacers (ITS1 and ITS2) among eight taxa of the Mimulus guttatus species complex

The internal transcribed spacer region (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA sequence and the intervening 5.8S region were sequenced from three individuals in each of eight taxa of the Mimulus guttatus species complex. Three discrete variants, or "types," of ITS sequences were found, among which 30%-40% of sites differed, compared with 1%-2% within types. Dot plots indicate that these types were not related by conspicuous rearrangements or inversions. More than one ITS type was often found in the same taxon, and two of three ITS types span species boundaries, indicating their presence prior to speciation. These ITS sequences showed essentially no positional homology with the nearest sequenced relative, tomato. In contrast, the 5.8S region was relatively unvaried, with 8 of 162 sites varied in the sample among all eight taxa. The phylogeny inferred by the most common ITS sequence type, rooted by the two other ITS types, agreed with isozymes in showing the distinctness of M. nudatus, M. laciniatus, and M. tilingii from the other five taxa.      

Antimicrobial activity of lactic acid bacteria isolated from goat's milk and artisanal cheeses: characteristics of a bacteriocin produced by Lactobacillus curvatus IFPL105

A total of 203 lactic acid bacteria isolated from raw goat's milk and artisanal cheese were tested for antibacterial activity. Only two strains of Lactococcus lactis , one strain of Enterococcus faecalis and one strain of Lactobacillus curvatus were shown to produce a bacteriocin-like substance. Lactobacillus curvatus IFPL105 produced a heat-stable bacteriocin, which was hydrolysed by α-chymotrypsin, proteinase K and pancreatin and exhibited a broad spectrum of inhibitory activity. The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes. Curing experiments resulted in variants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of the parental strain and the bacteriocin-negative variants indicated that a plasmid of about 46 kbp may be involved in bacteriocin production and immunity to this antibacterial compound.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

A comparison of measurements of intracellular Ca by Ca electrode and optical indicators

Squid giant axons were injected with aequorin or arsenazo III and impaled with a Ca-sensing electrode. The light output of aequorin or the spectrophotometer output when measuring arsenazo was compared with the voltage output of the electrode when the squid axon was depolarized with high-K solutions, when the seawater was made Na-free, or when the axon was tetanized for several minutes. The results from these treatments were that the optical response rose (as much as 50-fold) with all treatments known to increase Ca entry, while the electrode remained unaffected by these treatments. If axons previously subjected to Ca load are treated with electron-transport poisons such as CN, it is known that [Ca]_i rises after a time necessary to deplete ATP stores. In such axons one expects a rise of [Ca]_i in axoplasm which does not necessarily have to be uniform although the source of such Ca is the mitochondria and these are uniformly distributed in axoplasm. Under conditions of CN application, the optical signals from aequorin or arsenazo and Ca electrode output do rise together when [Ca]_i is high, but there is a region of [Ca]_i concentration where aequorin light output or arsenazo absorbance rises while electrode output does not. Axons not loaded with Ca but injected with apyrase and vanadate have mitochondria that still retain some Ca and this can be released by CN in a truly uniform manner. The results show that such a release (which is small) can be readily measured with aequorin, but again the Ca electrode is insensitive to such [Ca]_i change.     

Magnesium content and net fluxes in squid giant axons

The Mg content of axons freshly dissected from living specimens of the tropical squid Doryteuthis plei was determined by atomic absorption spectroscopy to be 4.2 +/- 0.2 mmol/kg axoplasm. The axon's ability to maintain this physiological content of total intracellular Mg([Mg]i) was studied. Mgi was shown to be a linear function of Mgo when Mgo of incubating fluid was varied between 0 and 250 mM. When Mgo = 15 mM, Mgi was found to be the same in incubated fibers as in fibers freshly dissected. Mgi levels were unaffected by depolarization of the membrane by high Ko. Stimulation resulted in an extra influx of Mg of 0.05 pmol/(cm2 . impulse) when Mgo = 55 mM. Mgi was found to be a complicated function of the concentration of extracellular Na or Li (Xo), which was substituted for Tris. With 385 mM Lio the Mgi level was found to be 2.5-fold larger than the level observed with 385 mM Nao after incubation for 3 h. The function relating Mgo to Xo was qualitatively unaffected in axons poisoned with the mitochondrial uncoupler carbonyl cyanide, p-trifluorome-thoxy-phenylhydrazone (FCCP) and the inhibitor of glycolysis, iodoacetic acid (IAA); the absolute levels of Mgi, however, were some 30% higher in the poisoned axons at all [X]o explored. 2 h incubation of axons in a 333 mM Mg, 40 mM Li solution increased Mgi 3.5-fold in control axons and 5-fold in poisoned axons. These Mg-loaded axons were able to recover physiological levels of Mgi with a half-time of 3-5 h only if kept in a solution which contained Na (220 mM) regardless of whether the axons had been inhibited with FCCP + IAA. Therefore, it may be concluded that the physiological Mgi concentration can be maintained by the Na electrochemical gradient, even when the axon is metabolically poisoned.     

Calcium content and net fluxes in squid giant axons

Axons freshly dissected from living specimens of the tropical squid Dorytheutis plei have a calcium content of 68 mumol/kg of axoplasm. Fibers stimulated at 100 impulses/s in 100 mM Ca seawater increase their Ca content by 150 mumol/kg.min; axons placed in 3 Ca (choline) seawater increase their Ca content by 12 mumol/kg.min. Axons loaded with 0.2--1.5 mmol Ca/kg of axoplasm extruded Ca with a half time of 15--30 min when allowed to recover in 3 Ca (Na) seawater. The half time for recovery of loaded axons poisoned with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and iodoacetic acid (IAA) is about the same as control axons. Axons placed in 40 mM Na choline seawater (to reduce chemical gradient for Na) or in 40 mM Na, 410 mM K seawater to reduce the electrochemical gradient for Na to near zero either fail to lose previously loaded Ca or gain further Ca.