β-Glucosidase from the cellulolytic system of Alternaria alternata autolyzed cultures

Abstract A β-glucosidase from centrifugated autolyzed cultures of Alternaria alternata has been purified 71 times by Sephadex G-200, CM-Biogel A and DEAE-Biogel A successively. The enzyme is a glycoprotein with 16% sugar and a M _r of 160 000, formed by two subunits of 60 000 and 80 000. The enzyme has optimum pH of 5 units and optimum reaction temperature of 50°C, being stable in a pH range of 3–8 and 0 to 60°C. The enzyme hydrolyzes different substrates showing maximum affinity and maximum hydrolysis velocity on cellobiose. The β-glucosidase is inhibited by gluconolactone but not by 10 mM glucose.     

DNA-methyltransferase activities in Streptomyces antibioticus

Abstract To quantitate ammonia production by the intestinal flora, ammonia levels in arterial blood and the venous effluent of the small and large bowel of conventional, selectively decontaminated, germ-free and gnotobiotic rats were measured.
When the anaerobic flora was removed by decontamination a significant decrease in ammonia levels was observed in the effluent of both the small and large intestine. Decontamination of aerobic flora did not result in depression of ammonia production. Gnotobiotic rats colonised with an anaerobic flora or with a mixed aerobic and anaerobic flora, showed a slight increase in ammonia levels. No increase in ammonia production was observed when rats were colonised with aerobic flora. These results indicate that the Enterobacteriaceae were not responsible for ammonia generation.
The increase in ammonia levels after colonisation with anaerobic or mixed anaerobic/aerobic flora did not completely restore ammonia levels, despite reaching bacterial counts which were comparable to those in conventional rats. This may be explained by the limited number of species with which the rats were colonized. The finding that aerobic flora does not significantly contribute to ammonia production suggests that neomycin, known to be exclusively effective against aerobic flora, must have other effects to explain improvement of hepatic encephalopathy.     

Early myocardial deformation changes associated to isolated obesity: a study based on 3D-wall motion tracking analysis

Obesity is considered as a strong risk factor for cardiovascular morbidity and mortality. 3D-wall motion tracking echocardiography (3D-WMT) provides information regarding different parameters of left ventricular (LV) myocardial deformation. Our aim was to assess the presence of early myocardial deformation abnormalities in nonselected obese children free from other cardiovascular risk factors. Thirty consecutive nonselected obese children and 42 healthy volunteer children were enrolled. None of them had any cardiovascular risk factor. Every subject underwent a 2D-echo examination and a 3D-WMT study. Mean age was 13.9 ± 2.56 and 13.25 ± 2.68 years in the nonobese and obese groups, respectively (59.7% and 40.3% male). Statistically significant differences were found for: interventricular septum thickness, LV posterior wall thickness, LV end-diastolic volume, LV end-systolic volume, left atrium volume, LV mass, and lateral annulus peak velocity. Regarding the results obtained by 3D-WMT assessment, all the evaluated parameters were statistically significantly different between the two groups. When the influence of obesity on the different echocardiographic variables was evaluated by means of multivariate logistic regression analysis, the strongest relationship with obesity was found for LV average circumferential strain (β-coefficient: 0.74; r(2): 0.55; P: 0.003). Thus, obesity cardiomyopathy is associated not only with structural cardiac changes, but also with myocardial deformation changes. Furthermore, this association occurs as early as in the childhood and it is independent from any other cardiovascular risk factor. The most related parameter to obesity is LV circumferential strain.     

PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides

Contamination of small-grain cereals with the fungal species Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti is an important source of trichothecenes, Zearalenone and other mycotoxins which cause serious diseases in human and animals. Additionally, these species contribute to Fusarium Head Blight, a disease which produces important losses in cereal yield. Early detection and control of these Fusarium species is crucial to prevent toxins entering the food chain and a useful tool in disease management practices. We describe the development of specific PCR assays to F. graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti using DNA from pure fungal cultures as well as from naturally infected wheat seeds, using in this case a rapid and easy protocol for DNA isolation. The specific primers were designed on the basis of IGS sequences (Intergenic Spacer of rDNA), a multicopy region in the genome that permits to enhance the sensitivity of the assay in comparison with PCR assays based on single-copy sequences.     

The African swine fever virus dynein-binding protein p54 induces infected cell apoptosis

A specific interaction of ASFV p54 protein with 8 kDa light chain cytoplasmic dynein (DLC8) has been previously characterized and this interaction is critical during virus internalization and transport to factory sites. During early phases of infection, the virus induces the initiation of apoptosis triggering activation of caspase-9 and -3. To analyze the role of the structural protein p54 in apoptosis, transient expression experiments of p54 in Vero cells were carried out which resulted in effector caspase-3 activation and apoptosis. Interestingly, p54 mutants, lacking the 13 aa dynein-binding motif lose caspase activation ability and pro-death function of p54. This is the first reported ASFV protein which induces apoptosis.     

A new nuclear protease with cathepsin L properties is present in HeLa and Caco‐2 cells

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell‐cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH‐protease) participates in male chromatin remodeling and in cell‐cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38–42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco‐2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non‐proliferative cells. J. Cell. Biochem. 111: 1099–1106, 2010. © 2010 Wiley‐Liss, Inc.     

Small Peptide Inhibitors Disrupt a High-Affinity Interaction between Cytoplasmic Dynein and a Viral Cargo Protein

Several viruses target the microtubular motor system in early stages of the viral life cycle. African swine fever virus (ASFV) protein p54 hijacks the microtubule-dependent transport by interaction with a dynein light chain (DYNLL1/DLC8). This was shown to be a high-affinity interaction, and the residues gradually disappearing were mapped on DLC8 to define a putative p54 binding surface by nuclear magnetic resonance (NMR) spectroscopy. The potential of short peptides targeting the binding domain to disrupt this high-affinity protein-protein interaction was assayed, and a short peptide sequence was shown to bind and compete with viral protein binding to dynein. Given the complexity and number of proteins involved in cellular transport, the prevention of this viral-DLC8 interaction might not be relevant for successful viral infection. Thus, we tested the capacity of these peptides to interfere with viral infection by disrupting dynein interaction with viral p54. Using this approach, we report on short peptides that inhibit viral growth.To enter the host cell, a virus must cross several barriers to reach the nucleus. Many viruses hijack the microtubular network to be transported along the cytoplasm (7, 18). Dynein is a microtubular motor protein, part of a large macromolecular complex called the microtubular motor complex. Dynein is involved in early stages of the viral life cycle of diverse infections, the first stage being the intracellular transport of the incoming virus along microtubules. Once transported throughout the cytosol, the virus rapidly gains the perinuclear area or the nucleus, where virus replication takes place. The disruption of microtubules or microtubular motor dynein function impairs the transport of a number of viruses; however, the intrinsic mechanism of this transport is unclear. Also, it has not been firmly established whether there is a common mechanism by which these viruses hijack a component of the microtubular motor complex for this purpose (7). A direct interaction between a given viral protein and cytoplasmic dynein for transport has been reported for HIV, herpes simplex virus, African swine fever virus (ASFV), and rabies virus (4, 14, 22, 25). In adenoviruses, a direct interaction of the viral capsid hexon subunit with cytoplasmic dynein has been described recently (5).One of these viruses, ASFV, which is a large DNA virus, enters the cell by dynamin- and clathrin-dependent endocytosis (12), and its infectivity is dependent on the acidification of the endosome. ASFV protein p54, a major protein of virion membranes, interacts with the light-chain dynein of 8 kDa (DLC8), which allows the transport of the virus to the perinuclear area (4), in a region called the microtubular organizing center (MTOC). In this zone, the virus starts replication in the viral factory, a secluded compartment where newly formed virions assemble (11, 13). By binding DLC8, the virus masters intracellular transport to ensure successful infection. However, due to the complexity of the system, the mechanism of this interaction is still elusive.A variety of names have been used for the subunits of the cytoplasmic dynein complex. A new classification for mammalian cytoplasmic dynein subunit genes based on their phylogenetic relationships has been reported in which the DLC8 gene was named DYNLL1 (26).Light dynein chains are responsible for direct cargo binding in the cell, but how do they select so many different cargos? It is not known whether the mode and site of binding is the same for viral proteins and physiological cargos. Within these multimeric complexes, there are a number of molecules that theoretically could interact with a given viral protein. However, to date viral proteins have been described to bind only light or intermediate dynein chains, such as DLC8 and TcTex1 (4, 5, 8). A candidate viral protein would bind one of the DLC binding domains, which in DLC8 are located between the two dimers of the DLC8 molecule (LysXThrThr). Here, we analyzed this interaction between a viral protein and DLC8 in an attempt to elucidate its requirements and relevance for viral infection.To determine whether this interaction is crucial for viral replication or whether it is just one of a number of alternatives for the virus-host interplay, we analyzed the capacity of a set of inhibitor peptides targeting a determined binding domain of the DLC8 molecule to interfere with viral infection by disrupting dynein interaction with viral p54.     

Cathepsin L inhibitor I blocks mitotic chromosomes decondensation during cleavage cell cycles of sea urchin embryos

We have previously reported that sperm histones (SpH) degradation after fertilization is catalyzed by a cystein-protease (SpH-protease). Its inhibition blocks the degradation of SpH in vivo and also aborts sea urchin development at the initial embryonic cell cycles. It remains unknown if this effect is a consequence of the persistence of SpH on zygotic chromatin, or if this protease is involved per-se in the progression of the embryonic cell cycles. To discriminate among these two options we have inhibited this protease at a time when male chromatin remodeling was completed and the embryos were engaged in the second cell cycle of the cleavage divisions. The role of this enzyme in cell cycle was initially analyzed by immuno-inhibiting its SpH degrading activity in one of the two blastomeres after the initial cleavage division, while the other blastomere was used as a control. We found that in the blastomere injected with the anti-SpH-protease antibodies the cytokinesis was arrested, the chromatin failed to decondense after mitosis and BrdU incorporation into DNA was blocked. Since the N-terminal sequence and the SpH protease was homologous to the cathepsin L (Cat L) family of proteases, we subsequently investigated if the deleterious effect of the inhibition of this protease is related to its Cat L activity. In this context we analyzed the effect of Cat L inhibitor I (Z-Phe-Phe-CH(2)F) on embryonic development. We found that the addition of 100 uM of this inhibitor to the embryos harvested at the time of the initial cleavage division (80 min p.i.) mimics perfectly the effects of the immuno-inhibition of this enzyme obtained by microinjecting the anti-SpH-protease antibodies. Taken together these results indicate that the activity of this protease is required for embryonic cell cycle progression. Interestingly, we observed that when this protease was inhibited the chromatin decondensation after mitosis was abolished indicating that the inhibition of this enzyme affects chromosomes decondensation after mitosis.