Dual Insecticidal Effects of Adenanthera pavonina Kunitz-Type Inhibitor on Plodia interpunctella is Mediated by Digestive Enzymes Inhibition and Chitin-Binding Properties.

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions.

A New Salt-Tolerant Thermostable Cellulase from a Marine Bacillus sp. Strain.

A salt-tolerant cellulase secreted by a marine Bacillus sp. SR22 strain with wide resistance to temperature and pH was purified and characterized. Its approximate mass was 37 kDa. The endoglucanase, named as Bc22Cel, was purified by ammonium sulfate precipitation, gel filtration chromatography, and extraction from the gel after non-reducing sodium dodecyl sufate-polyacrylamide gel electrophoresis. The optimal pH value and temperature of Bc22Cel were 6.5 and 60°C, respectively. The purified Bc22Cel showed a considerable halophilic property, being able to maintain more than 70% of residual activity even when pre-incubated with 1.5 M NaCl for 1 h. Kinetic analysis of the purified enzyme showed the Km and Vmax to be 0.704 mg/ml and 29.85 µmol·ml-1·min-1, respectively. Taken together, the present data indicate Bc22Cel as a potential and useful candidate for industrial applications, such as the bioconversion of sugarcane bagasse to its derivatives.

Inhibitory effects of a Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests' digestive proteinases.

Pithecellobium dumosum is a tree belonging to the Mimosoideae subfamily that presents various previously characterized Kunitz-type inhibitors. The present study provides a novel Kunitz-trypsin inhibitor isoform purified from P. dumosum seeds. Purification procedure was performed by TCA precipitation followed by a trypsin-Sepharose chromatography and a further reversed-phase HPLC. Purified inhibitor (PdKI-4) showed enhanced inhibitory activity against bovine trypsin and chymotrypsin. Furthermore, PdKI-4 showed remarkable inhibitory activity against serine proteases from the coleopterans Callosobruchus maculatus and Zabrotes subfasciatus, and the lepidopterans Alabama argillacea and Telchin licus. However, PdKI-4 was unable to inhibit porcine pancreatic elastase, pineapple bromelain and Carica papaya papain. SDS-PAGE showed that PdKI-4 consisted of a single polypeptide chain with molecular mass of 21 kDa. Kinetic studies demonstrated that PdKI-4 is probably a competitive inhibitor with a Ki value of 5.7 × 10(-10) M for bovine trypsin. PdKI-4 also showed higher stability over a wide range of temperature (37-100 °C) and pH (2-12). N-termini sequence was obtained by Edman degradation showing higher identity with other Mimosoideae subfamily Kunitz-type inhibitor members. In summary, data here reported indicate the biotechnological potential of PdKI-4 for development of products against insect-pests.

Hypocitraturia: a risk factor for reduced bone mineral density in idiopathic hypercalciuria?

UNLABELLED: The association between idiopathic hypercalciuria (IH) and reduced bone mineral density (BMD) has been described in adults and children. Frequently, hypocitraturia (HC) is an associated condition. To determine the effect that HC may have on bone metabolism of these patients, we studied 88 children with IH at diagnosis, divided into the following groups: group 1-44 (50%) patients with associated HC; group 2-44 (50%) patients without HC; group 3 (29 subjects), a healthy control group. Urinary and blood electrolytes, as long as urinary N-telopeptide, were measured. Lumbar spine (L2-L4) and femoral neck bone mineral density (BMD) and bone mineral content (BMC) were measured by dual energy X-ray absorptiometry. There was no difference in age between the three groups (P=0.80), but weight, height, body mass index, and bone age were lower (P<0.01) and serum intact parathyroid hormone (iPTH) was higher (P<0.05) in group 1 than in groups 2 and 3. N-telopeptide, measured in urine, did not differ between groups. The following bone densitometry parameters: lumbar spine BMC, BMC adjusted for height (BMCh), BMC adjusted for width of vertebrae (BMCw) and BMD, as well as femoral neck BMD, were significantly lower in group 1 than in groups 2 and 3 (P<0.01). When we corrected densitometry parameters for height, BMC was lower in group 1 and not in group 2 when compared with controls. CONCLUSIONS: Children with IH and associated HC may have a higher risk of bone mass loss and consequent osteopenia. Further studies are needed to assess the role that hypocitraturia may have in this form of bone disease.

Bone alterations in children with idiopathic hypercalciuria at the time of diagnosis.

Some children with idiopathic hypercalciuria (IH) develop bone alterations at some stage of the disease. The aims of this study were to evaluate bone mass in 88 children with IH (G1) at the time of diagnosis and to compare the findings with data for a control group of 29 normal children (G2). Kidney and bone metabolism markers were measured in both groups, and bone densitometry was performed. Serum alkaline phosphatase, intact parathyroid hormone, urinary calcium and uric acid were significantly higher in G1, whereas urinary volume and urinary citrate excretion were lower. The following densitometric parameters were significantly lower in G1: (1) lumbar spine (L(2)-L(4)) bone mineral density (BMD), bone mineral content (BMC), BMC corrected for height and for width of the vertebra, volumetric BMD (BMDvol), and Z score; (2) whole-body BMD; (3) femoral neck BMD. Lumbar spine BMDvol was reduced (osteopenia) in 35% of the patients compared with G2. N telopeptide, a urinary marker of bone resorption, was significantly higher in G1 than in G2, and was negatively correlated with lumbar spine BMD and BMDvol. Children with urinary lithiasis or idiopathic hyperuricosuria associated with IH showed no significant differences in bone metabolism compared with children without these associations. We conclude that (1) there is an altered bone metabolism in IH, with osteopenia already present at diagnosis in 35% of the patients; (2) N telopeptide is one of the most useful markers of bone alterations in IH, especially at an early stage of the disease; (3) investigation of bone metabolism is necessary in IH to prevent future serious consequences such as osteoporosis and bone fractures.