Discovery of a new isomannide-based peptidomimetic synthetized by Ugi multicomponent reaction as human tissue kallikrein 1 inhibitor

Human kallikrein 1 (KLK1) is the most extensively studied member of this family and plays a major role in inflammation processes. From Ugi multicomponent reactions, isomannide-based peptidomimetic 10 and 13 where synthesized and showed low micromolar values of IC₅₀ for KLK1 The most active compound (10) presented competitive mechanism, with three structural modifications important to interact with active site residues which corroborates its KLK1 inhibition. Finally, the most active compound also showed good ADMET profile, which indicates compound 10 as a potential hit in the search for new KLK1 inhibitors with low side effects.     

Lack of GDAP1 Induces Neuronal Calcium and Mitochondrial Defects in a Knockout Mouse Model of Charcot-Marie-Tooth Neuropathy

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca²⁺ inflow through store-operated Ca²⁺ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca²⁺, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria–endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.     

3-Methylcrotonylglycine Disrupts Mitochondrial Energy Homeostasis and Inhibits Synaptic Na+,K+-ATPase Activity in Brain of Young Rats

Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an inherited metabolic disease biochemically characterized by accumulation and high urinary excretion of 3-methylcrotonylglycine (3MCG), and also of 3-hydroisovalerate in lesser amounts. Affected patients usually have neurologic dysfunction, brain abnormalities and cardiomyopathy, whose pathogenesis is still unknown. The present study investigated the in vitro effects of 3MCG on important parameters of energy metabolism, including CO₂ production from labeled acetate, enzyme activities of the citric acid cycle, as well as of the respiratory chain complexes I–IV (oxidative phosphorylation), creatine kinase (intracellular ATP transfer), and synaptic Na⁺,K⁺-ATPase (neurotransmission) in brain cortex of young rats. 3MCG significantly reduced CO₂ production, implying that this compound compromises citric acid cycle activity. Furthermore, 3MCG diminished the activities of complex II-III of the respiratory chain, mitochondrial creatine kinase and synaptic membrane Na⁺,K⁺-ATPase. Furthermore, antioxidants were able to attenuate or fully prevent the inhibitory effect of 3MCG on creatine kinase and synaptic membrane Na⁺,K⁺-ATPase activities. We also observed that lipid peroxidation was elicited by 3MCG, suggesting the involvement of free radicals on 3MCG-induced effects. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production, creatine kinase for intracellular energy transfer, and Na⁺,K⁺-ATPase for the maintenance of the cell membrane potential, the present data indicate that 3MCG potentially impairs mitochondrial brain energy homeostasis and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by 3-methylcrotonyl-CoA carboxylase deficiency.     

New antimalarial indolone-N-oxides, generating radical species, destabilize the host cell membrane at early stages of Plasmodium falciparum growth: role of band 3 tyrosine phosphorylation

Although indolone-N-oxide (INODs) genereting long-lived radicals possess antiplasmodial activity in the low-nanomolar range, little is known about their mechanism of action. To explore the molecular basis of INOD activity, we screened for changes in INOD-treated malaria-infected erythrocytes (Pf-RBCs) using a proteomics approach. At early parasite maturation stages, treatment with INODs at their IC(50) concentrations induced a marked tyrosine phosphorylation of the erythrocyte membrane protein band 3, whereas no effect was observed in control RBCs. After INOD treatment of Pf-RBCs we also observed: (i) accelerated formation of membrane aggregates containing hyperphosphorylated band 3, Syk kinase, and denatured hemoglobin; (ii) dose-dependent release of microvesicles containing the membrane aggregates; (iii) reduction in band 3 phosphorylation, Pf-RBC vesiculation, and antimalarial effect of INODs upon addition of Syk kinase inhibitors; and (iv) correlation between the IC(50) and the INOD concentrations required to induce band 3 phosphorylation and vesiculation. Together with previous data demonstrating that tyrosine phosphorylation of oxidized band 3 promotes its dissociation from the cytoskeleton, these results suggest that INODs cause a profound destabilization of the Pf-RBC membrane through a mechanism apparently triggered by the activation of a redox signaling pathway rather than direct oxidative damage.     

Use of Bacillus thuringiensis Toxins for Control of the Cotton Pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae)

Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 μg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 μg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 μg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. ¹²⁵I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.     

Detection and characterization of protease secreted by the plant pathogen Xylella fastidiosa

Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a copolymerized substrate. Three major protein bands were produced by the citrus strain with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9,713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) activity gel indicated that the protease of Xylella fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30 degrees C with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and beta-1,3-glucanase activities were also detected in cultures of Xylella fastidiosa (citrus). From these results, it is suggested that proteases produced by strains of Xylella fastidiosa from citrus and grape, belong to the serine- and metallo-protease group, respectively.     

Medium-chain fatty acids accumulating in MCAD deficiency elicit lipid and protein oxidative damage and decrease non-enzymatic antioxidant defenses in rat brain

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent disorder of fatty acid oxidation with a similar prevalence to that of phenylketonuria. Affected patients present tissue accumulation of the medium-chain fatty acids octanoate (OA), decanoate (DA) and cis-4-decenoate. Clinical presentation is characterized by neurological symptoms, such as convulsions and lethargy that may develop into coma and sudden death. The aim of the present work was to investigate the in vitro effect of OA and DA, the metabolites that predominantly accumulate in MCADD, on oxidative stress parameters in rat cerebral cortex homogenates. It was first verified that both DA and OA significantly increased chemiluminescence and thiobarbituric acid-reactive species levels (lipoperoxidation) and decreased the non-enzymatic antioxidant defenses, measured by the decreased total antioxidant capacity. DA also enhanced carbonyl content and oxidation of sulfhydryl groups (protein damage) and decreased reduced glutathione (GSH) levels. We also verified that DA-induced GSH decrease and sulfhydryl oxidation were not observed when cytosolic preparations (membrane-free supernatants) were used, suggesting a mitochondrial mechanism for these actions. Our present data show that the medium-chain fatty acids DA and OA that most accumulate in MCADD cause oxidative stress in rat brain. It is therefore presumed that this pathomechanism may be involved in the pathophysiology of the neurologic symptoms manifested by patients affected by MCADD.     

Clarified cashew apple juice as alternative raw material for biosurfactant production by Bacillus subtilis in a batch bioreactor

Clarified cashew apple juice was evaluated as carbon source for surfactin production by Bacillus subtilis LAMI005 isolated from the tank of chlorination at the Wastewater Treatment Plant on Campus do Pici (WWTP-PICI) in the Federal University of Ceará, Brazil. The highest surfactin concentration using clarified cashew apple juice (CCAJ) supplemented with mineral medium (MM-CCAJ) was 123 mg/L, achieved after 48 h of fermentation. Almost 2-fold less than the amount produced using mineral medium supplemented with 10 g/L of glucose and 8.7 g/L of fructose (MM-GF). However, critical micelle concentration of the biosurfactants produced using MM-CCAJ was 2.5-fold lower than the one produced using MM-GF, which indicates it is a more efficient biosurfactant. Surface tension decreased from 38.50 ± 0.0 to 29.00 ± 0.0 dyne/cm when B. subtilis was grown on MM-CCAJ media (24.68% of reduction on surface tension) and remained constant up to 72 h. Emulsification index was 51.15 and 66.70% using soybean oil and kerosene, respectively. Surfactin produced in MM-CCAJ showed an emulsifying activity of, respectively, 1.75 and 2.3 U when n-hexadecane or soybean oil was tested. However, when mineral medium supplemented with 10 g/L of glucose (MM-G) was used an emulsifying activity of 2.0 and 1.75 U, with n-hexadecane and soybean oil, respectively, was obtained. These results indicate that it is feasible to produce surfactin from CCAJ, a renewable and low-cost carbon source.