A biochemical logic gate using an enzyme and its inhibitor. Part II: The logic gate

Enzyme-Based Logic Gates (ENLOGs) are key components in bio-molecular systems for information processing. This report and the previous one in this series address the characterization of two bio-molecular switching elements, namely the alpha-chymotrypsin (alphaCT) derivative p-phenylazobenzoyl-alpha-chymotrypsin (PABalphaCT) and its inhibitor (proflavine), as well as their assembly into a logic gate.The experimental output of the proposed system is expressed in terms of enzymic activity and this was translated into logic output (i.e. "1" or "0") relative to a predetermined threshold value. We have found that an univalent link exists between the dominant isomers of PABalphaCT (cis or trans), the dominant form of either acridine (proflavine) or acridan and the logic output of the system. Thus, of all possible combinations, only the trans-PABalphaCT and the acridan lead to an enzymic activity that can be defined as logic output "1". The system operates under the rules of Boolean algebra and performs as an "AND" logic gate.     

Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques

Summary Ultrastructural post-embedding immuno-gold techniques were applied to the supraoptic nucleus and the neurohypophysis of mice and rats. The primary antibodies were three different monoclonal antineurophysins, used in protein A-gold and immunoglobulin-gold procedures. Conventional plastic embedding as well as hydrophilic media (L.R. White) were used; non-osmicated and osmicated tissues were immunolabeled; sodium metaperiodate oxidation was used, but was not essential for immunolabeling.Vasopressinergic and oxytocinergic NSGs were identified by the specific immunoreactivity of their respective neurophysins on adjacent thin sections, and by sequential double labeling on the same thin section using two different antibodies associated with gold probes of different diameters. The immunoidentification indicates that vasopressin NSGs can additionally be differentiated as larger, with more electron-dense matrix, and susceptible to damage by sodium metaperiodate.The only organelles consistently labeled were neurosecretory granules (NSGs), either intact or within lysosomal configurations. Some lysosomal dense bodies were immunoreactive even when discrete NSGs were no longer morphologically recognisable within them. Labeled NSGs were located within neuronal cell bodies, along axonal shafts and within axonal swellings and endings; occasionally immunoreactive NSGs were observed within synaptic boutons. Labeling intensity was semi-quantitatively gauged by counting gold particles in relation to numbers of NSGs per axonal varicosity.The precise localisation achieved with particulate immunogold labeling surpasses that previously obtained with diffuse electron-dense immunoreaction products.     

Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358

BackgroundSevere leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis.Methodology/Principal findingsMale Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6–7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression.Conclusion/SignificanceThis study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis.     

The role of indigenous bacterial and fungal soil populations in the biodegradation of crude oil in a desert soil

The biodegradation capacity of indigenous microbial populations was examined in a desert soil contaminated with crude oil. To evaluate biodegradation, soil samples supplemented with 5, 10 or 20% (w/w) of crude oil were incubated for 90 days at 30 °C. The effect of augmentation of the soil with vermiculite (50% v/v) as a bulking agent providing increased surface/volume ratio and improved soil aeration was also tested. Maximal biodegradation (91%) was obtained in soil containing the highest concentration of crude oil (20%) and supplemented with vermiculite; only 74% of the oil was degraded in samples containing the same level of crude oil but lacking vermiculite. Gas chromatograms of distilled fractions of crude oil extracted from the soil before and after incubation demonstrated that most of the light and part of the intermediate weight fractions initially present in the oil extracts could not be detected after incubation. Monitoring of microbial population densities revealed an initial decline in bacterial viable counts after exposure to oil, presumably as a result of the crude oil’s toxicity. This decline was followed by a steep recovery in microbial population density, then by a moderate increase that persisted until the end of incubation. By contrast, the inhibitory effect of crude oil on the fungal population was minimal. Furthermore, the overall increased growth response of the fungal population, at all three levels of contamination, was about one order of magnitude higher than that of the bacterial population.     

New perspectives in plastic biodegradation

During the past 50 years new plastic materials, in various applications, have gradually replaced the traditional metal, wood, leather materials. Ironically, the most preferred property of plastics--durability--exerts also the major environmental threat. Recycling has practically failed to provide a safe solution for disposal of plastic waste (only 5% out of 1 trillion plastic bags, annually produced in the US alone, are being recycled). Since the most utilized plastic is polyethylene (PE; ca. 140 million tons/year), any reduction in the accumulation of PE waste alone would have a major impact on the overall reduction of the plastic waste in the environment. Since PE is considered to be practically inert, efforts were made to isolate unique microorganisms capable of utilizing synthetic polymers. Recent data showed that biodegradation of plastic waste with selected microbial strains became a viable solution.     

Mutations disrupting selenocysteine formation cause progressive cerebello-cerebral atrophy

The essential micronutrient selenium is found in proteins as selenocysteine (Sec), the only genetically encoded amino acid whose biosynthesis occurs on its cognate tRNA in humans. In the final step of selenocysteine formation, the essential enzyme SepSecS catalyzes the conversion of Sep-tRNA to Sec-tRNA. We demonstrate that SepSecS mutations cause autosomal-recessive progressive cerebellocerebral atrophy (PCCA) in Jews of Iraqi and Moroccan ancestry. Both founder mutations, common in these two populations, disrupt the sole route to the biosynthesis of the 21st amino acid, Sec, and thus to the generation of selenoproteins in humans.     

Ribosomal slowdown mediates translational arrest during cellular division

Global mRNA translation is transiently inhibited during cellular division. We demonstrate that mitotic cells contain heavy polysomes, but these are significantly less translationally active than polysomes in cycling cells. Several observations indicate that mitotic translational attenuation occurs during the elongation stage: (i) in cycling nonsynchronized cultures, only mitotic cells fail to assemble stress granules when treated with agents that inhibit translational initiation; (ii) mitotic cells contain fewer free 80S complexes, which are less sensitive to high salt disassembly; (iii) mitotic polysomes are more resistant to enforced disassembly using puromycin; and (iv) ribosome transit time increases during mitosis. Elongation slowdown guarantees that polysomes are retained even if initiation is inhibited at the same time. Stalling translating ribosomes during mitosis may protect mRNAs and allow rapid resumption of translation immediately upon entry into the G(1) phase.     

Metal-dependent anaerobic methane oxidation in marine sediment: Insights from marine settings and other systems

Anaerobic oxidation of methane(AOM) plays a crucial role in controlling global methane emission. This is a microbial process that relies on the reduction of external electron acceptors such as sulfate, nitrate/nitrite, and transient metal ions. In marine settings, the dominant electron acceptor for AOM is sulfate, while other known electron acceptors are transient metal ions such as iron and manganese oxides. Despite the AOM process coupled with sulfate reduction being relatively well characterized,researches on metal-dependent AOM process are few, and no microorganism has to date been identified as being responsible for this reaction in natural marine environments. In this review, geochemical evidences of metal-dependent AOM from sediment cores in various marine environments are summarized. Studies have showed that iron and manganese are reduced in accordance with methane oxidation in seeps or diffusive profiles below the methanogenesis zone. The potential biochemical basis and mechanisms for metal-dependent AOM processes are here presented and discussed. Future research will shed light on the microbes involved in this process and also on the molecular basis of the electron transfer between these microbes and metals in natural marine environments.