A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 15°C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with K_i values of modified savinases sixfold higher than the unmodified savinase. Modeling of small‐angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold‐active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme‐formulation. Biotechnol. Bioeng. 2009;103: 676–686. © 2009 Wiley Periodicals, Inc.     

Naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates: Potent, non-cytotoxic, antiapoptotic agents

Compounds containing a quinone moiety represent an important class of biologically active molecules that are widespread in nature, displaying anticancer, antibacterial, antimalarial, and fungicidal activities. In the course of designing 2,3-disubstituted-1,4-naphthoquinones derivatives as potential cysteine protease inhibitors, two naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates, 1a and 1b, were obtained. The antiapoptotic potential of 1a and 1b was then evaluated and compared to that of naphthoquinone 4. Primary rat hepatocytes were incubated with synthesized naphthoquinone derivatives and then exposed to the apoptotic stimulus camptothecin. Our results indicate that naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates 1a and 1b exerted a potent protective role in camptothecin-induced apoptosis in primary rat hepatocytes. Both 1a and 1b significantly increased cell viability, while reducing nuclear fragmentation, caspase-3, -8 and -9 activation, and cytochrome c release induced by camptothecin. In addition, 1a and 1b were shown to up-regulate Bcl-X_L, a pro-survival member of the Bcl-2 family of proteins, which modulates the mitochondrial pathway of apoptosis. Similar protective effects of quinone derivatives were seen in HuH-7 and PC12 cells incubated with distinct apoptotic stimuli, such as camptothecin, TGF-β1, or rotenone. Our results suggest that naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates 1a and 1b may act as potent, cytoprotective agents, through modulation of apoptotic pathways.     

Influence of recovery method and microbial contamination on the response to freezing–thawing in ibex (Capra pyrenaica) epididymal spermatozoa

The method of sperm recovery may influence the initial quality of sperm samples and their response to freezing–thawing. The aim of the present work was to compare two methods for collecting epididymal spermatozoa in order to improve the quality of recovered sperm and reduce possible contamination. Testes were obtained from 23 legally hunted, adult ibex males. The sperm mass of the right epididymis was collected by small longitudinal and transverse cuts made in the cauda epididymidis. The sperm mass of the left epididymis was collected by retrograde flushing from the vas deferens to the cauda epididymidis (using a cannula), employing a Tris, citric acid, glucose, egg yolk-based medium. The flushing method recovered more spermatozoa (P < 0.001) than the cutting method. After freezing–thawing, greater acrosomes damage (P < 0.001) and more morphological abnormalities (P < 0.05) were seen among the sperm cells recovered by the cutting method than among those obtained by retrograde flushing. The method of sperm recovery did not, however, influence the microbial contamination rate. In frozen–thawed samples that were microbially contaminated, motility was significantly reduced (P < 0.05) and membrane integrity tended to be poorer (P = 0.06). In conclusion, retrograde flushing is recommended for ibex sperm collection since it would appear that microbial contamination is no more of a problem than that encountered with the cutting method, while a larger number of sperm cells more resistant to freezing–thawing can be obtained.     

Variability in the behavioural responses of three generalist herbivores to the most abundant coumarin in Daphne laureola leaves

We evaluated the responses of three generalist herbivores (Lepidoptera: Noctuidae) to the most abundant coumarin in young leaves of spurge-laurel, Daphne laureola L. (Thymelaeaceae), a perennial shrub consistently fed upon by several noctuid species. Pseudenargia ulicis Staud and Noctua janthe Borkhausen are natural herbivores of the species in southeastern Spain, whereas Spodoptera littoralis Boisduval is a model species frequently used to address the antifeedant role of secondary compounds. Discrimination between control and coumarin-coated D. laureola leaves was investigated by short-term choice experiments. We found species-specific behavioural responses to the treatment: P. ulicis preferred the control leaves, whereas S. littoralis did not discriminate between leaf treatments, although it clearly avoided coumarin when incorporated into an artificial diet, and N. janthe preferred the coumarin-coated leaves. Furthermore, N. janthe larvae reduced consumption when the proportion of treated leaves ingested increased and consumption of S. littoralis larvae was also reduced in coumarin-containing diet under no-choice conditions. Our results highlight that different herbivore species feeding simultaneously on the same host plant respond differently to a single chemical defence compound, likely constraining a directional response of the plant to selection.     

Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from São Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.     

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.Cholinergic neurotransmission has key functions in life, as it regulates several central and peripheral nervous system outputs. Acetylcholine (ACh) is synthesized in the cytoplasm by the enzyme choline acetyltransferase (ChAT) (16). Choline supplied by the high-affinity choline transporter (CHT1) is required to maintain ACh synthesis (52). A lack of ChAT (4, 35) or the high-affinity choline transporter (21) in genetically modified mice is incompatible with life. ACh plays an important role in wiring the neuromuscular junction (NMJ) during development (38, 43). Embryonic synthesis of ACh is fundamental for the development of proper nerve-muscle patterning at the mammalian NMJ, as ChAT-null mice present aberrant nicotinic ACh receptor (nAChR) localization and increased motoneuron (MN) survival, axonal sprouting, and branching (4, 35).The vesicular ACh transporter (VAChT) exchanges cytoplasmic ACh for two vesicular protons (37, 41). Previously reported electrophysiological studies showed that quantal size is decreased by vesamicol, an inhibitor of VAChT, but only in nerve terminals that have been electrically stimulated (19, 59, 60, 63). VAChT overexpression in developing Xenopus MNs increases both the size and frequency of miniature-end-plate currents (54). In Caenorhabditis elegans, mutations in VAChT affect behavior (65). Moreover, a decrease in VAChT expression has functional consequences for mammals, as mutant mice with a 70% reduction in the expression levels of this transporter (VAChT knockdown [KD^HOM] mice) are myasthenic and have cognitive deficits (47). Hence, vesicular transport activity is rate limiting for neurotransmission “in vivo” (18, 47).Exocytosis of synaptic vesicle contents is the predominant mechanism for the regulated secretion of neurotransmitters (55). However, alternative mechanisms of secretion have been proposed (20, 56, 61). Quantal ACh release, comparable to that seen in developing nerve terminals, has been detected in myocytes and fibroblasts in culture, which presumably do not express VAChT (14, 24). More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64). The result suggests that the release of ACh during development is not dependent on VAChT, perhaps because it is nonvesicular or because vesicular storage can occur without VAChT.To test if the VAChT-independent secretion of ACh has any physiological role in the mammalian nervous system, we generated a mouse line in which the VAChT gene is deleted. These mice lack the stimulated release of ACh from synaptosomes, die after birth, and show several alterations in neuromuscular wiring consistent with a severe decrease in the cholinergic input to muscles during development. These experiments indicate that VAChT has an important role in maintaining activity-dependent ACh release that supports life and the correct patterning of innervation at the NMJ.     

Does the Future Affect the Present? The Effects of Future Weather on the Current Collection of Planted Crops and Wildlife in a Native Amazonian Society of Bolivia

Unlike neighboring disciplines, anthropology rarely studies how actual future events affect current behavior. Such studies could lay the groundwork for studies of ethno-forecasting. Psychologists argue that people forecast poorly, but some empirical work in cultural anthropology suggests that at least with weather, rural people might make reasonably accurate forecasts. Using data from a small-scale, pre-industrial rural society in the Bolivian Amazon, this study estimates the effects of future weather on the current collection of planted crops and wildlife. If actual future events affect current behavior, then this would suggest that people must forecast accurately. Longitudinal data covering 11 consecutive months (10/2002–8/2003, inclusive) from 311 women and 326 men ≥age 14 in 13 villages of a contemporary society of forager-farmers in Bolivia’s Amazon (Tsimane’) are used. Individual fixed-effect panel linear regressions are used to estimate the effect of future weather (mean hourly temperature and total daily rain) over the next 1–7 days from today on the probability of collecting wildlife (game, fish, and feral plants excluding firewood) and planted farm crops (annuals and perennials) today. Daily weather records come from a town next to the Tsimane’ territory and data on foraging and farming come from scans (behavioral spot observations) and surveys of study participants done during scans. Short-term future weather (≤3 days) affected the probability of collecting planted crops and wildlife today, although the effect was greater on the amount of planted crops harvested today than on the amount of wildlife collected today. Future weather beyond 3 days bore no significant association with the amount of planted crops harvested today nor with the amount of wildlife collected today. After controlling for future and past weather, today’s weather (mean hourly temperature, but not rain) affected the probability of collecting wildlife today, but today’s weather (temperature or rain) did not affect the probability of collecting planted crops today. The study supports prior work by anthropologists suggesting that rural people forecast accurately. If future weather affects the probability of harvesting planted crops and collecting wildlife today, then this suggests that Tsimane’ must forecast accurately. We discuss possible reasons for the finding. The study also supports growing evidence from rural areas of low-income nations that rural people tend to protect their food production and food consumption well against small idiosyncratic shocks or, in our case, against ordinary daily weather that is not extreme. However, the greater responsiveness of daily foraging output compared with daily farming output to today’s weather suggests that foraging might not protect food consumption as well as farming against adverse climate perturbations.     

The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum

The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.     

Differential localization of α’ and β subunits of protein kinase CK2 during rat spermatogenesis

Protein kinase CK2 is a serine/threonine kinase expressed in organisms from yeast to human and is composed of a catalytic subunit (α or α’) and a regulatory subunit (β) forming a holoenzyme with the possible subunit combinations α₂β₂, α’₂β₂, or αα’β₂. This kinase has been shown to be involved in embryonic development and gametogenesis. We have studied the expression of the CK2α’ and CK2β subunits during the first wave of spermatogenesis and in adult testis in the rat. Western blot analyses have demonstrated that both CK2α’ and CK2β are expressed in testes from birth to adulthood. A more detailed study of the protein localization of CK2α’ and CK2β by immunohistochemistry suggests that CK2α’ and CK2β are localized in the nuclei of Sertoli cells in 5-day-old rats, whereas they appear to have a cytoplasmic localization in older animals. In adult testes, CK2α’ and CK2β subunits are present in spermatocytes. Both subunits exhibit a similar expression pattern with the highest level in spermatocytes at stages VIII-XIV. Interestingly, CK2β is highly expressed in spermatogonia, whereas CK2α’ is barely detectable. Mature epididymal spermatozoa express CK2α’ in the acrosome and CK2β in the flagellum. This new evidence therefore indicates that protein kinase CK2 has a possible role at various stages during mammalian spermatogenesis, a process that involves proliferation, meiosis, apoptosis, and differentiation. CK2 might thus emerge as a new pivotal control enzyme at various levels in mammalian spermatogenesis.