Biochemical comparison of two proteolytic enzymes from Carica candamarcensis: Structural motifs underlying resistance to cystatin inhibition

The lattices of Carica candamarcensis and Carica papaya, members of the Caricaceae family, contain isoforms of cysteine proteinases that help protect these plants against injury. In a prior study, we fractionated 14 discrete proteinaceous components from C. candamarcensis, two of them displaying mitogenic activity in mammalian cells. In this study, we compared the kinetic parameters of one of the mitogenic proteinases (CMS2MS2) with one of the isoforms displaying the highest enzyme activity of this group (CMS1MS2). Both enzymes display a similar Km value with either BAPNA (Benzoyl-Arg-pNA) or PFLPNA (Pyr-Phe-Leu-pNA), but the kcat of CMS1MS2 is about 14-fold higher for BAPNA and 129-fold higher with PFLPNA. While both enzymes are inhibited by E-64 and iodoacetamide, chicken cystatin fully inhibits CMS1MS2, but scarcely affects activity of CMS2MS2. Based on the structure of these proteins and other enzymes from the Caricaceae family whose structures have been resolved, it is proposed that Arg¹⁸⁰ located in the cleft at the active site in CMS2MS2 is responsible for its resistance to cystatin.     

Pectinase production by a Brazilian thermophilic fungus <Emphasis Type=Italic>Thermomucor indicae-seudaticae</Emphasis> N31 in solid-state and submerged fermentation

Thermophilic organisms produce thermostable enzymes, which have a number of applications, justifying the interest in the isolation of new thermophilic strains and study of their enzymes. Thirty-four thermophilic and thermotolerant fungal strains were isolated from soil, organic compost, and an industrial waste pile based on their ability to grow at 45°C and in a liquid medium containing pectin as the only carbon source. Among these fungi, 50% were identified at the genus level as Thermomyces, Aspergillus, Monascus, Chaetomium, Neosartoria, Scopulariopsis, and Thermomucor. All isolated strains produced pectinase during solid-state fermentation (SSF). The highest polygalacturonase (PG) activity was obtained in the culture medium of thermophilic strain N31 identified as Thermomucor indicae-seudaticae. Under SSF conditions on media containing a mixture of wheat bran and orange bagasse (1 : 1) at 70% of initial moisture, this fungus produced the maximum of 120 U/ml of exo-PG, while in submerged fermentation (SmF) it produced 13.6 U/ml. The crude PG from SmF was more thermostable than that from SSF and exhibited higher stability in acidic pH.     

Effect of bacterial growth in the complexing capacity of a culture medium supplemented with cadmium(II)

The aim of this study was to evaluate complexing capacity (CC) accompanying microbial growth in a metal supplemented culture medium. A combined strategy of square wave anodic stripping voltammetry (SWASV) monitored titration and ion-exchange resins treatment (Chelex 100) has been applied. Culture medium, supplemented with Cd(II) in excess to ligands, was inoculated with an indigenous bacterial culture; total ligand concentration and stability constants were determined at different bacterial growth stages. As far as known, determination of CC in such conditions has not been reported (usually ligands in natural or wastewaters exceed metal concentration). HIDA, (N-(2-hydroxyethyl)iminodiacetic acid), was used as a model ligand to mimic soluble products derived from the resin treatment and bacterial metabolism. Ligand concentration, Lt (1.3 ± 0.1 μM), and the conditional stability constant, K′, (log K′ = 5.7 ± 0.2) were in good agreement with expected values (1.0 ± 0.1 μM and log K′ = 6.1). In the supplemented culture medium, total ligand concentration in the micromolar range (60–80 μM) and conditional stability constants (5.5 < log K′ < 6.5) were determined. Cd(II) complexes detected in the different stages of microbial growth are labile from an electrochemical point of view. Results were compared to the case of Cd(II) non-supplemented broth.     

Observations on the invading gastropod <Emphasis Type=Italic>Rapana venosa</Emphasis> in Punta del Este,Maldonado Bay,Uruguay

We report on the first sightings of the invasive Rapa Whelk Rapana venosa in Maldonado Bay (Punta del Este Harbor and Gorriti Island) using in vivo, underwater observations and video surveys. The species was first detected in the Río de la Plata (Uruguay and Argentina) in 1999, and by 2004 it had extended its local distribution to Punta del Este at the eastern boundary of the estuary. Observations performed by SCUBA diving showed that R. venosa is preying on native mussels Mytilus edulis and Brachidontes spp., and that formerly abundant mussel beds are being seriously depleted due to a combination of human extraction, habitat deterioration and predation by the Rapa Whelk.     

Anti‐cancer therapy: senescence is the new black

Cellular senescence is a natural safeguard against cancer. Pharmacologically, it can be induced by drugs that inhibit the CDK4/6 kinases such as palbociclib, but the exact mechanism has never been dissected. Recent research by Miettinen et al ( 2018 ) reveals that senescence induced by this class of drugs is mediated by proteasome hyper‐activation.     

Antifungal properties of Canavalia ensiformis urease and derived peptides

Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO₂. These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli. The antifungal activity of ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H⁺ and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to ureases. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10⁻⁶–10⁻⁷ M.     

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.     

No Association between Low Birth Weight and Cardiovascular Risk Factors in Early Adulthood: Evidence from S?o Paulo,Brazil

Background

A growing literature suggests that low birth weight increases the risk of poor health outcomes in adulthood. We tested this hypothesis among young adults living in São Paulo State, Brazil.

Methods and Findings

To identify the effects of low birth weight on young adulthood outcomes, a medical assessment of 297 individuals born between 1977 and 1989 was conducted at a primary care unit in São Paulo State, Brazil. We analyzed body mass index (BMI), waist-hip ratio, blood pressure, fasting glucose and total cholesterol levels using linear and logistic regressions. Low birth was negatively associated with BMI (β = −2.0, 95% CI: −3.69, −0.27, p = 0.02), fasting glucose levels (β = −1.9, 95% CI: −3.9, −0.07, p = 0.05), waist-hip ratio (β = −0.03, 95% CI: −0.07, −0.01, p = 0.10), systolic blood pressure (β = −3.32, 95% CI: −7.60, 0.96, p = 0.12), and total cholesterol levels (β = −3.19, 95% CI: −16.43, 10.05, p = 0.636). Low birth weight was also associated with lower odds of young adults being overweight and obese, but neither association was statistically significant. Weight gain in the first 12 months of life was associated with higher adult BMI (β = 0.79, 95% CI: −0.0455, 1.623, p = 0.064) and blood pressure (β = 2.79, 95% CI: 0.22, 5.35, p = 0.034). No associations were found between low birth weight and early life (catch-up) growth.

Conclusions

Low birth weight was not associated with poor health outcomes among young adults in Brazil. These results appear inconsistent with the original Barker hypothesis, but will need to be corroborated in larger samples with longer follow-ups to allow a more general evaluation of the validity of the hypothesis in low and middle income countries.