Helicoverpa armigera cadherin fragment enhances Cry1Ac insecticidal activity by facilitating toxin-oligomer formation

The interaction between Bacillus thuringiensis insecticidal crystal protein Cry1A and cadherin receptors in lepidopteran insects induces toxin oligomerization, which is essential for membrane insertion and mediates Cry1A toxicity. It has been reported that Manduca sexta cadherin fragment CR12-MPED and Anopheles gambiae cadherin fragment CR11-MPED enhance the insecticidal activity of Cry1Ab and Cry4Ba to certain lepidopteran and dipteran larvae species, respectively. This study reports that a Helicoverpa armigera cadherin fragment (HaCad1) containing its toxin binding region, expressed in Escherichia coli, enhanced Cry1Ac activity against H. armigera larvae. A binding assay showed that HaCad1 was able to bind to Cry1Ac in vitro and that this event did not block toxin binding to the brush border membrane microvilli prepared from H. armigera. When the residues ¹⁴²³GVLSLNFQ¹⁴³⁰ were deleted from the fragment, the subsequent mutation peptide lost its ability to bind Cry1Ac and the toxicity enhancement was also significantly reduced. Oligomerization tests showed that HaCad1 facilitates the formation of a 250-kDa oligomer of Cry1Ac-activated toxin in the midgut fluid environment. Oligomer formation was dependent upon the toxin binding to HaCad1, which was also necessary for the HaCad1-mediated enhancement effect. Our discovery reveals a novel strategy to enhance insecticidal activity or to overcome the resistance of insects to B. thuringiensis toxin-based biopesticides and transgenic crops.     

Infection Status of Hospitalized Diarrheal Patients with Gastrointestinal Protozoa, Bacteria, and Viruses in the Republic of Korea

To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged ≤ 5 years was significantly higher, while C. perfringens among bacterial infection was higher for ≥ 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea.     

Bilateral similarity function: A novel and universal method for similarity analysis of biological sequences

Bilateral similarity function is designed for analyzing the similarities of biological sequences such as DNA, RNA secondary structure or protein in this paper. The defined function can perform comprehensive comparison between sequences remarkably well, both in terms of the Hamming distance of two compared sequences and the corresponding location difference. Compared with the existing methods for similarity analysis, the examination of similarities/dissimilarities illustrates that the proposed method with the computational complexity of O(N) is effective for these three kinds of biological sequences, and bears the universality for them.     

Birth intervention and non-maternal infant-handling during parturition in a nonhuman primate

Direct intervention in infant delivery by non-parturient individuals is a rare phenomenon in nonhuman primates. In contrast, birth assistance by other individuals, or the practice of midwifery, is universal among human societies and generally believed to be a behavior unique to our species. It has been proposed that the enlarged head of the human fetus and the relatively narrow birth canal constrained by bipedalism has made human parturition more difficult than in nonhuman primates, and these anatomic challenges have led to the rotation of the fetus in the birth canal and an occiput anterior (i.e., backward-facing) orientation of emergence. These characteristics have hindered the mother’s ability to self-assist the delivery of the infant, therefore necessitating assistance by other individuals or midwives for successful birth. Here we report the first high-definition video recordings of birth intervention behavior in a wild nonhuman primate, the white-headed langur (Trachypithecus leucocephalus). We observed that while a primiparous female gave birth to an infant in an occiput posterior (i.e., forward-facing) orientation, a multiparous female intervened in the delivery by manually pulling the infant out of the birth canal and cared for it in the following hours. Our finding shows extensive social interactions throughout parturition, and presents an unequivocal case of non-maternal intervention with infant birth in a nonhuman primate.     

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin

Trifluoperazine (TFP; Stelazine?) is an antagonist of calmodulin (CaM), an essential regulator of calcium‐dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca²⁺)₄‐CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM‐kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca²⁺)₄‐CaM and explore differential effects on the N‐ and C‐domains of CaM, stoichiometric TFP titrations of CaM were monitored by ¹⁵N‐HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca²⁺)₄‐CaM. In both cases, the preferred site was in the C‐domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP‐binding sites in apo CaM appeared distinct from those in (Ca²⁺)₄‐CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ‐motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N‐domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed,” “semi‐open,” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca²⁺)₄‐CaM, needs to be considered a potential target of drug action. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Hydrolysis of filter-paper cellulose to glucose by two recombinant endogenous glycosyl hydrolases of Coptotermes formosanus

Abstract Genes encoding for glycosyl hydrolases (GH) in multiple families were recovered from an expression sequence tag library of Coptotermes formosanus, a xylophagous lower termite species. Functional analyses of these genes not only shed light on the mechanisms the insect employs to successfully use cellulosic materials as energy sources, which may serve as strategic targets for designing molecular-based bio-pesticides, but also enrich discoveries of new cellulolytic enzymes for conversion of biomass into biofuel. Our study demonstrated that cellulose could be converted to glucose by two recombinant endogenous glycosyl hydrolases (endo-β-1,4 glucanase in GH9 and β-glucosidase in GH1). While the former cleaved cellulose to cellobiose and cellotriose, the resulting simple cellodextrins were digested to glucose. Both of the Escherichia coli-expressed recombinant proteins showed properties that could be incorporated in a glucose-based ethanol production program.     

Stereoscopic Analysis of Optic Nerve Head Parameters in Primary Open Angle Glaucoma: The Glaucoma Stereo Analysis Study

PurposeThe Glaucoma Stereo Analysis Study (GSAS), a cross sectional multicenter collaborative study, used a stereo fundus camera to assess various morphological parameters of the optic nerve head (ONH) in glaucoma patients and investigated the relationships between these parameters and patient characteristics.ResultsPatient characteristics included refractive error of −3.38±3.75 diopters, intraocular pressure (IOP) of 13.6±2.6 mmHg, and visual field mean deviation (MD) of −4.71±3.26 dB. Representative ONH parameters included a horizontal disc width of 1.66±0.28 mm, vertical disc width of 1.86±0.23 mm, disc area of 2.42±0.63 mm², cup area of 1.45±0.57 mm², and cup volume of 0.31±0.22 mm³. Correlation analysis revealed significant negative associations between vertical cup-to-disc ratio (0.82±0.08) and MD (r = −0.40, P<0.01) and between disc tilt angle (10.5±12.5 degrees) and refractive error (r = −0.36, P<0.01). Seventy-five percent of the eyes had a positive value for rim decentering (0.30±0.42), indicating that rim thinning manifested more often as an inferior lesion than a superior lesion.ConclusionWe used stereoscopic analysis to establish a database of ONH parameters, which may facilitate future studies of glaucomatous changes in ONH morphology.     

Susceptibility to cell death induced by blockade of MAPK pathway in human colorectal cancer cells carrying Ras mutations is dependent on p53 status

Constitutive activation of mitogen-activated protein kinase (MAPK) pathway is implicated in a variety of human malignancies especially those that carry Ras mutations and is currently exploited as a cancer therapeutic target. The variability of response by cancer cells to the inhibition of the Ras/MAPK pathway both in vivo and in vitro, however, suggests that the genetic background of the tumor cell may modulate the effectiveness of this directed therapeutic. In a panel of colorectal cancer cell lines that carry Ras mutations and have constitutively active MEK/MAPK, we found that inhibition of the MAPK upstream kinase MEK by the small molecular MEK inhibitor U0126 induced cell death only in p53 wild-type cells. By contrast, p53-deficient cells were not affected by blocking the MEK/MAPK pathway. Using isogenic colon cancer cell lines and RNA interference, we show that loss of p53 significantly reduces MAPK phosphorylation and renders cells resistant to U0126 treatment. These findings reveal a critical role for p53 in MAPK-driven cell survival and place p53 upstream in the control cascade of MAPK activity. The therapeutic implication of these observations is that MAPK inhibitors will be most beneficial as a therapeutic agent in p53 normal colon cancers where constitutively active MAPK resulting from a Ras mutation is required for cell survival.     

An approach to the residence time distribution for stochastic multi-compartment models

Stochastic compartmental models are widely used in modeling processes such as drug kinetics in biological systems. This paper considers the distribution of the residence times for stochastic multi-compartment models, especially systems with non-exponential lifetime distributions. The paper first derives the moment generating function of the bivariate residence time distribution for the two-compartment model with general lifetimes and approximates the density of the residence time using the saddlepoint approximation. Then, it extends the distributional approach to the residence time for multi-compartment semi-Markov models combining the cofactor rule for a single destination and the analytic approach to the two-compartment model. This approach provides a complete specification of the residence time distribution based on the moment generating function and thus facilitates an easier calculation of high-order moments than the approach using the coefficient matrix. Applications to drug kinetics demonstrate the simplicity and usefulness of this approach.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.