Trypsin inhibitors in xoconostle seeds (Opuntia joconostle Weber.)

Seed proteins recovered after heating a seed extract from Opuntia joconostle Weber (xoconostle, an acid cactus pear) were screened for different biochemical activities, detecting only trypsin-like inhibitory activity. Two trypsin-like inhibitor forms from seeds were separated by RP-HPLC and partially sequenced and characterized as an enriched mixture. They were evaluated for inhibition on several serine proteinases, but only trypsin-like inhibition was detected by the inhibitor extract. The two isolated forms, OjTI 1 and OjTI 2 showed low molecular weights of 4.26 and 4.17 kDa as determined by mass spectrometry. An enriched inhibitory fraction showed a high thermal stability by retaining the activity after heating the sample for 1 h at 90 °C, as well as after heating for at 120 °C under 1 kg/cm² for 15 min at different pH values. Partial sequence of the two forms was determined by mass spectrometry indicating that they were similar and after alignments analysis they showed the highest similarity with the trypsin inhibitor from O. streptacantha and to a lesser extent to other trypsin inhibitors of the MEROPS database families. The inhibitory spectrum was evaluated against several digestive enzymes from pests and beneficial insects from several taxonomic orders.     

Electroacupuncture and Curcumin Promote Oxidative Balance and Motor Function Recovery in Rats Following Traumatic Spinal Cord Injury

Neurochemical Research - Spinal cord injury (SCI) is a condition that puts the patient’s life at risk in the acute phase and, during the chronic stage, results in permanent deficits in motor,...     

Stimulation of the Po-shen and Shen-hun scalp-acupuncture bands modifies levels of inhibitory and excitatory amino acids in the immature rat brain

Objective

In the present study, the effect of stimulation of the Po-shen and Shen-hun scalp-acupuncture bands on tissue amino acid concentrations in several brain regions in awake and pentobarbital-sedated immature rats was evaluated.

Materials and methods

Sprague–Dawley rats (aged 15 days) were organized in four groups of at least eight animals: control groups received saline solution 0.9% or sodium pentobarbital at 30 mg/kg dosage via intraperitoneal. Experimental groups received saline solution or sodium pentobarbital plus stimulation in Po-shen and Shen-hun scalp-acupuncture bands for one continuous hour during 10 sessions by using scalp-acupuncture.

Results

As compared to rats receiving saline solution, scalp-acupuncture produced significant changes in amino acid concentrations, depending on the analyzed region, as follows: in inhibitory amino acids, a GABA increase was observed in amygdala and hippocampus (491 and 184%, respectively), but a decrease in the substantia nigra (80%); glycine showed decrease in all the analyzed regions, except for an increase in brainstem(78%); glutamine presented an increase in hippocampus and cortex (42 and 149%, respectively). In the case of excitatory amino acids, glutamate decreased in all the analyzed regions; whereas aspartate decreased in substantia nigra and brainstem (77.08 and 35%, correspondingly) but increased in hippocampus and cortex (32 and 54%, respectively). The combined treatment of scalp-acupuncture and a GABAergic depressant drug like pentobarbital resulted in almost all changes induced in amino acids for scalp-acupuncture alone being significantly reverted.

Conclusion

Stimulation of the Po-shen and Shen-hun scalp-acupuncture bands by using scalp-acupuncture alone might produce depressant activity by changes in amino acids, but the combination with a GABAergic tranquilizer like sodium pentobarbital can interfere with this response.     

Tentative Identification of Phytochemicals and Antioxidant Activities during Fruit-Ripening on <i>Chamaedorea radicalis</i> Mart.

This work aims to determine the phytochemical characterization of the pericarp of Chamaedorea radicalis Mart. fruit as a non-timber product with potential to obtain phytochemicals with potential applications in the industry. Fruit from C. radicalis were grouped in four ripening stages named as S1, S2, S3 and S4, according to maturity; S1 the most unripe stage and S4 the completely ripe stage. Determinations of total phenolic compounds, free radical scavenging activities and total flavonoid contents using spectrophotometric methods were done. Also, the tentative identification of phytochemicals during fruit ripening was done using UPLC-MS-MS. Total phenolic compound (TPC) content ranged from 7.24 to 12.53 mg gallic acid equivalents per gram of fresh weight (mg GAE/ g FW). Total flavonoids (TF) contents ranged from 0.163 to 0.23 mg of quercetin equivalents per g FW (mg QE/g FW). Free radical scavenging activity against DPPH and ABTS radicals varied from 40.80 to 53.68 and from 22.29 to 37.76 mmol Trolox equivalents g FW (mmol TE/g FW), respectively. Antioxidant assay in vitro by FRAP (ferric reducing antioxidant power) method showed that S3 was the highest level with antioxidant power while S4 was the lowest with Red ripeness stage showed the lowest contents for all determinations. Mass spectrometry allowed detection of 26 compounds, including phenolics, alkaloids and saponins. Afzelin, Kaempferol 3-neohesperidoside and the four saponins identified were present in all ripeness stages. Preliminary phytochemical identi- fication and the spectrophotometric determinations showed that the pericarp of C. radicalis presented antioxidants and compounds related to alkaloids, phenolics and saponins. The presence and abundance of each phytochemical regarding each ripeness stage should be considered.     

Production and characterization of fungal <Emphasis Type="Italic">β</Emphasis>-glucosidase and bacterial cellulases by tobacco chloroplast transformation

The high capacity of the chloroplast genome to integrate and express transgenes at high levels makes transplastomic technology a good option for overexpressing proteins of interest. This report presents the stable expression of β-glucosidase (bgl1 gene) from Aspergillus niger and two cellulases (celA and celB genes) from Thermotoga neapolitana into the chloroplast genome of tobacco. The pES6, pHM4, pHM5 and pHM6 vectors were derived from the pES4 plasmid containing bgl1, celA-celB, celA and celB synthetic genes, respectively. All of the genes were flanked by a synthetic rrn16 promoter and the 3′UTR from rbcL gene. The integration of the genes into intergenic regions rrn16 and 3′rps12 of the inverted repeats was confirmed by Southern blot analysis. Stable expression and processing of monocistronic mRNA were confirmed by Northern blot analysis, and protein functionality was analysed via enzymatic activity assay. The recombinant enzymes exhibited high enzymatic activity at pH 5 (β-glucosidase: 30.45 U mg^?1 of TSP, celA-celB 58 U mg^?1 of TSP, celA 49.10 U mg^?1 of TSP and celB 48.72 U mg^?1 of TSP). In addition, β-glucosidase exhibited high activity at 40 °C, whereas cellulases type A (celA) and type B (celB) showed high activity at 65 °C. NtpES6, NtpHM5 and NtpHM6 plants showed a similar phenotype compared with the wild type plants; however, NtpHM4 plants presented an abnormal phenotype with variegated leaves. This study, demonstrated that hydrolytic genes such as bgl1, celA and celB could be integrated and expressed correctly in the chloroplast genome. This work provides new information on methods and strategies for the expression of hydrolytic enzymes that are potentially useful for biotechnological applications using transplastomic plants.     

Characterization of a highly stable trypsin-like proteinase inhibitor from the seeds of Opuntia streptacantha (O. streptacantha Lemaire)

A trypsin inhibitor from Opuntia streptacantha Lemaire (Prickly pear) seeds was purified and characterized. Of several proteases tested, this inhibitor showed specificity to trypsin-like enzymes. The major inhibitor present in these seeds showed distinctive characteristics, most notably a low molecular weight of 4.19 kDa, as determined by MALDI TOF, and an unusually high thermal stability, retaining most of the activity after heating the sample 1 h to 120 °C with a pressure of 1 kg/cm². Its complete amino acid sequence was obtained through mass spectrometry, this establishing presence a blocked N-terminal region. When comparing its sequence in the MEROPS database for peptidases and peptidase inhibitors, it showed 34.48% identity with a serine-proteinase inhibitor from the I15 family.