Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR<Subscript>1</Subscript>

Background  

Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR₁), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR₁ action.     

Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

Single-surgeon experience of 87 buccal mucosal graft harvests

Although buccal mucosa has been the preferred graft material for use in the reconstruction of anterior urethral strictures for approximately 20 years, the plastic surgery literature is lacking in studies describing and evaluating this technique. The authors sought to report their experience with the technique of buccal mucosa graft harvest for use in urethral reconstruction. The authors identified 87 consecutive patients who underwent buccal mucosal graft harvesting during a 15-year period from 1995 to 2010. All donor sites were closed primarily. Preoperatively, a specialty-specific evaluation was performed by plastic surgeons and urologists. Postoperative donor-site morbidity was followed clinically. Eighty-seven patients underwent buccal harvesting. A single buccal graft was harvested in 75 patients (86.2 percent) and bilateral grafts were harvested in 12 patients (13.8 percent). The median patient age of the patients was 42 years (range, 16 to 78 years). There were no major intraoperative or postoperative complications. Minor complications included mild discomfort, numbness, cheek swelling, and restriction of oral movement, all of which resolved by the 3-month postoperative visit. The ability to eat and drink postoperatively was diminished during only the first 24 to 48 hours in all patients. Our series provides a 1-year follow-up in a substantial cohort looking at clinical outcomes after buccal harvesting. These results confirm the safety and efficacy of buccal mucosal harvesting to provide an effective and cosmetically superior option for urethral reconstruction. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.     

Expression in Bacillus subtilis of the gene for human urogastrone using synthetic ribosome binding sites

Summary A chemically synthesised gene coding for human urogastrone which was earlier cloned in E. coli (Smith et al. 1982) has now been cloned into expression vectors for Bacillus subtilis Two types of constructs have been made, one giving production of methionylurogastrone and the other giving rise to a methionyl-urogastrone- galactosidase fusion polypeptide facilitating quantification of expression levels.The ribosome binding sites used in the expression plasmids are synthetically made oligonucleotides residing on short restriction fragments to allow easy replacement by other ribosome binding sites.Using shuttle vectors and constitutive promoters from Bacillus phages 105 and SPP1, we were able to detect levels of expression amounting to a few thousand molecules per cell during logarithmic growth in both E. coli and B. subtilis.     

Effect of alveolar hypoxia on pulmonary fluid filtration in in situ dog lungs

We have studied the effect of alveolar hypoxia on fluid filtration characteristics of the pulmonary microcirculation in an in situ left upper lobe preparation with near static flow conditions (20 ml/min). In six dogs (group 1), rate of edema formation (delta W/delta t, where W is weight and t is time) was assessed over a wide range of vascular pressures under two inspired O2 fraction (FIO2) conditions (0.95 and 0.0 with 5% CO2-balance N2 in both cases). delta W/delta t was plotted against vascular pressure, and the best-fit linear regression was obtained. There was no significant difference (paired t test) in either threshold pressure for edema formation [18.3 +/- 1.8 and 17.1 +/- 1.2 (SE) mmHg, respectively] or the slopes (0.067 +/- 0.008 and 0.073 +/- 0.017 g.min-1. mmHg-1.100g-1, respectively). In another seven dogs (group 2), delta W/delta t was obtained at a constant vascular pressure of 40 mmHg under four FIO2 conditions (0.95, 0.21, 0.05, and 0.0, with 5% CO2-balance N2). Delta W/delta t for the four conditions averaged 0.60 +/- 0.11, 0.61 +/- 0.11, 0.61 +/- 0.10, and 0.61 +/- 0.10 (SE) g.min-1.mmHg-1.100g-1, respectively. No significant differences (ANOVA for repeated measures) were noted. We conclude that alveolar hypoxia does not alter the threshold for edema formation or delta W/delta t at a given microvascular pressure.     

Electroantennogram responses of the oriental fruit fly, Dacus dorsalis, to a spectrum of alcohol and aldehyde plant volatiles

Electroantennograms (EAGs) were recorded from unmated, laboratory-reared, male and female oriental fruit flies, Dacus dorsalis, in response to a range of between C₁ and C₁₂ carbon chain-length saturated and unaturated aliphatic alcohols and aldehydes, most all of which are known host-plant volatiles. Only two of the 35 compounds tested elicited significantly larger EAGs from female than male antennae. For the two functional-group series tested, aldehydes elicited responses greater than or equal to the responses to the alcohols. In general, the unsaturated alcohols did not elicit responses significantly different from the saturated alcohols. However, the unsaturated aldehydes, (E)-2-hexenal and 10-undecenal, elicited larger amplitude EAGs than their saturated analogs. EAGs were significantly greater for a particular carbon chain-length, with responsiveness to primary alcohols peaking at C₆ and aldehydes peaking at C₇. The (E)-2- monoenic alcohols peaked at C₆, while the (E)-3-alcohols plateaued between C₅ and C₈. The greatest EAG responses of all compounds tested were elicited by the saturated and unsaturated C₆ alcohols and aldehydes which are constitutents of the general green-leaf volatile complex that emanates from most plants. The potential adapative benefit of selective sensitivity to green-leaf volatiles is discussed in regards to foraging behaviors of oriental fruit flies.

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Résumé Des électroantennogrammes (EAG) ont enregistré les réponses, en élevages de femelles et mâles vierges de Dacus dorsalis, à une gamme de chaînes de carbones de C₁ à C₁₂ saturés et non-saturés d'alcools aliphatiques et d'aldéhydes, dont beaucoup sont connus comme substances volatiles des végétaux. Seulement 2 des 35 composés examinés ont provoqué des EAG significativement plus importants chez les femelles que chez les mâles. Pour les séries des deux groupes fonctionnels examinés, les aldéhydes ont provoqué des réponses supérieures ou égales aux alcools. En général, les réponses aux alcools nonsaturés n'étaient pas significativement différentes des réponses aux alcools saturés. Cependant, les aldéhydes non-saturés, (E)-2-hexénal et 10-undécénal, ont induit des EAG de plus grande ampleur que leurs analogues saturés. Les EAG étaient significativement les plus importants pour une chaîne de longueur particulière, la réponse aux alcools primaires culminant en C₆ et les aldéhydes en C₇. Les alcools monoéniques (E)-2- culminaient en C₆, tandis que les alcools (E)-3- étaient étales entre C₅ et C₈. Les EAG les plus importants ont été obtenus pour tous les composés examinés avec les alcools et aldéhydes en C₆ qui appartiennent à l'odeur verte complexe émise par beaucoup de plantes. Le bénéfice adaptatif potentiel de la sensibilité sélective à l'odeur verte des feuilles est examinée en fonction du comportement de prospection de D. dorsalis.

     

Refolding of sepharose-bound trypsinogen with disulfide 179 to 203 reduced and carboxymethylated

Disulfide 179 to 203 of native bovine trypsin was reduced with sodium borohydride and converted to the S-carboxymethyl derivative. The modified zymogen was attached to CNBr-activated Sepharose, and the resulting immobilized protein was used in refolding studies. The fully reduced protein was kept at 35°, at pH 8.5, under aerobic conditions, in a mixture of reduced and oxidized glutathione, until the sulfhydryl groups were reoxidized. A maximum yield of 55% was found for the regeneration of S-(carboxymethyl)₂-trypsinogen, and the activated product, S-(carboxymethyl)₂-trypsin, reacted with an active site reagent and gave the expected specific activity toward a typical trypsin substrate. Apparently, the refolding of immobilized S-(carboxymethyl)₂-trypsinogen regenerated the native structure of trypsinogen even though one of the six disulfides could no longer be formed.     

Cyclic AMP blocks cell growth through Raf-1-dependent and Raf-1-independent mechanisms

It is widely accepted that cyclic AMP (cAMP) can block cell growth by phosphorylating Raf-1 on serine 43 and inhibiting signaling to extracellular signal-regulated protein kinase. We show that the suppression of Raf-1 by cAMP is considerably more complex than previously reported. When cellular cAMP is elevated, Raf-1 is phosphorylated on three residues (S43, S233, and S259), which work independently to block Raf-1. Both Ras-dependent and Ras-independent processes are disrupted. However, when cAMP-insensitive versions of Raf-1 are expressed in NIH 3T3 cells, their growth is still strongly suppressed when cAMP is elevated. Thus, although Raf-1 appears to be an important cAMP target, other pathways are also targeted by cAMP, providing alternative mechanisms that lead to suppression of cell growth.     

Crystal structures of two potent nonamidine inhibitors bound to factor Xa

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.