Proteinases of Streptomyces fradiae. II. Specificity.

It has been shown that extracellular proteinases synthesized by a keratinolytic strain of S. fradiae are characterized by diversified activity in the decomposition of both proteins and synthetic substrates. Among the six proteinases isolated, apart from the ones dominating and having relatively low specificity, there are two enzymes characterized by narrow catalytic abilities--extremely similar to those of trypsin. These proteinases intensively degraded all the trypsin substrates studied, but they were inactive or showed slight activity toward others. They were also highly sensitive to such specific inhibitors of trypsin as TLCK, SBTI and TIO.     

Structure and stability of transposon 5-mediated cointegrates

We have determined the structure of a set of independently derived, Tn5-mediated cointegrates and examined the stability of several examples. A variety of cointegrate structures was found, including those mediated by the entire compound transposon, and those mediated by a single flanking IS50 element, which was always IS50-R, and never IS50-L. IS50-R but not IS50-L is reported to code for a protein(s) required for transposition. This finding confirms that IS50-L is relatively inactive and suggests that the active transposition protein(s) acts largely in cis on IS50-R. Another class of cointegrate was created by inverse transposition of Tn5 (using the inside ends of the flanking elements). In addition, we found an unexpectedly large set of cointegrates, in which the joint between the two plasmids was not adjacent to the transposon. All cointegrates analysed were found to be stable. This suggests that Tn5, unlike the transposon Tn3, does not transpose via an obligate cointegrate intermediate. This finding is compared to previous results with Tn5 and Tn9, and is discussed in terms of current models of transposition.     

Genetic analysis of transpositions in the lac region of Escherichia coli

The lac region of Escherichia coli, carried on an F′ lacproB episome, was used as a target for the transposition of several transposable elements. Tn9 shows a preferential integration (by a factor of 50) into a region extending from the end of the Z gene through the Y gene. Throughout the remainder of the lacI, Z and Y genes one other short region, located in the middle of the I gene, is favored for integration. Within these favored regions many different integration points are evident. Inspection of the DNA sequence for the I and Y genes, and parts of the Z gene, shows a strong correlation between A + T richness and regions of preferential integration. Tn5 insertions follow a similar pattern, although with less preference; whereas Tn10 insertions (provided by T. J. Foster), also favor the Y gene and the end of Z, but are distributed among fewer integration points. Most of the Tn3 insertions into the episome are accompanied by a nearby or adjacent deletion.     

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.     

Chicoric Acid Is an Antioxidant Molecule That Stimulates AMP Kinase Pathway in L6 Myotubes and Extends Lifespan in Caenorhabditis elegans

Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5–100 μM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.     

From spring sources to springbrook: Changes in environmental characteristics and benthic fauna

Spatial patterns in macroinvertebrate communities and some abiotic factors were examined in three rheocrene springs and their springbrooks (Kraków-Cz?stochowa Upland, southern Poland). The mean discharge of particular springs ranged from 5 to 11 L s^?1, and its annual fluctuations were small. Water temperature was very stable at all sampling sites. In the eucrenon the number of benthic taxa was the smallest (9–14 determined to the family level), but the densities were the highest (approx. 14000 ind. m^?2). The biggest changes in macroinvertebrate composition were observed in the modified hypocrenon, which is an artificial pond. The lowest number of taxa were found in a natural, short springbrook with a nondiversified bottom substrate. The density of crenophilic taxa (Drusus trifidus, Dugesia gonocephala, Elmidae) diminished along the springbrooks, while the opposite trend was observed for ubiquitous taxa (some Oligochaeta, Asellus aquaticus and Chironomidae). Even in a very short natural springbrook (30 m), Drusus trifidus, the only species of Trichoptera found in the springs discussed here, goes through the entire development cycle. The strongest influence of a big river was observed at the outflow of one of the natural springbrooks, where the highest number of riverine oligochaete species were found. The benthic fauna of the springs studied here differed from that found in other springs in this area — the absence of the typical crenophilic species Bithynella austriaca (Gastropoda) and the presence of Gianus aquedulcis (Oligochaeta) may indicate the autonomy of the spring fauna in the Mstów area, possibly resulting from the postglacial geomorphological formation of this region or differences in habitat conditions.     

Voltage and calcium use the same molecular determinants to inactivate calcium channels

During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha1C Ca2+ channel) are similarly influenced by Ca2+ channel beta subunits. The same molecular determinants of the beta subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.     

Immunohistochemical localization of androgen receptor and aromatase in the ovary of the pregnant pig

The distribution of androgen receptor (AR) and cytochrome P450 aromatase was investigated in paraffin sections of pregnant pig ovary. Ovarian follicles and corpora lutea were isolated from ovaries obtained on Days 10, 18, 32, 71 and 90 post coitum (p.c.). Androgen receptor was localized in the nuclei of granulosa cells of follicles of various sizes. In addition, some follicles demonstrated staining in the nuclei of theca interna cells. Stroma cells also exhibited a positive immunostaining. At early and mid pregnancy (up to Day 71) AR was expressed in the nuclei of luteal cells. Corpora lutea of Day 71 showed mainly cytoplasmic staining while on Day 90 almost all luteal cells showed staining exclusively in the cytoplasm. Immuno-staining for the presence of cytochrome P450 aromatase was very faint in all investigated ovarian structures. The results could suggest that the process of androgen aromatization plays a negligible role in the ovary of the pregnant pig.     

Neurotrophic effects of PACAP in the cerebellar cortex

In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human.