Effects of activation of μ-, κ<Subscript>1</Subscript>-, δ<Subscript>1</Subscript>-, δ<Subscript>2</Subscript>-, and ORL<Subscript>1</Subscript>-receptors on heart resistance to the pathogenic action of delayed ischemia and reperfusion

Different subtypes of opioid receptors (OR) were activated in rats in vivo to study the activation effect on the heart’s resistance to ischemia and reperfusion. It has been established that administration of deltorphin II, a selective δ₂-OR agonist, lowered the infarct size/area at risk index (IS/AAR) by 23%. Naltrexone, naloxone methiodide (an OR inhibitor not penetrating the blood-brain barrier (BBB)), and naltriben (δ₂-antagonist) eliminated the cardioprotective effect of deltorphin II, while BNTX (a δ₁-antagonist) produced no effect on the cardioprotective action of the δ₂-agonist. The infarct-reducing effect of deltorphin II was eliminated by administration of chelerythrine (a protein kinase C (PKC) inhibitor), glibenclamide (a K_ATP-channels inhibitor), and 5-hydroxydecanoate (a mitochondrial K_ATP-channel blocker). Administration of other opioids did not reduce the IS/AAR index. It has been established that all the deltorphins manifest antiarrhythmic potency. Other opioids do not produce any effect on the incidence of arrhythmia occurrences. The antiarrhythmic effect of deltorphin II was eliminated by preliminary administration of naltrexone, naloxone methiodide, and naltriben, but BNTX did not affect the δ₂-agonist’s anti-arrhythmic effect. The preliminary administration of chelerythrine, a PKC inhibitor, eliminated the δ₂ agonist’s antiarrhythmic action. However, glibenclamide and 5-hydroxydecanoate did not alter the antiarrhythmic effect by deltorphin II. Therefore, activation of the peripheral δ₂-ORs reduces the infarct size and prevents the onset of arrhythmias. The antiarrhythmic effect of the δ₂-OR stimulation is mediated by activating PKC and opening the mitochondrial K_ATP-channels. PKC participates in the antiarrhythmic effect of the δ₂-OR activation, but this effect does not depend on the condition of K_ATP-channels.     

Crystal structure of the γ-cyclodextrin n-propanol inclusion complex; Correlation of α-, β-, γ-cyclodextrin geometries

Crystals of the hydrated n-propanol inclusion complex of γ-cyclodextrin (γ-CD; cyclo-octaamylose) have space group P4, a = b = 23.759(7), c = 23.069(7)Å and six quarter γ-CD per asymmetric unit. The structure was solved by YZARC and refined to R = 14% using 6300 X-ray counter data. The γ-CD are stacked, n-propanol (not located) occupies the channel-type cavity and 27 water sites populate interstices between stacks. Within the stacks γ-CD are arranged head-to-head as well as head-to-tail and H-bonded with O(2), O(3), O(6) hydroxyls. In the series α-,β-,γ-CD, angles C(1′)-O(4)-C(4) reduce from 119°-117.7°-112.6°, virtual O(4′)?O(4) distances increase 4.23-4.39-4.48 Å. intramolecular H-bonding distances O(2)?O(3) between adjacent glucoses, 3.00 Å in α-CD are wider than ～2.83 Å in β- and γ-CD, indicating a greater flexibility of the former.     

SDF2L1, a Component of the Endoplasmic Reticulum Chaperone Complex, Differentially Interacts with ��-, ��-, and ��-Defensin Propeptides

Mammalian defensins are cationic antimicrobial peptides that play a central role in host innate immunity and as regulators of acquired immunity. In animals, three structural defensin subfamilies, designated as α, β, and θ, have been characterized, each possessing a distinctive tridisulfide motif. Mature α- and β-defensins are produced by simple proteolytic processing of their prepropeptide precursors. In contrast, the macrocyclic θ-defensins are formed by the head-to-tail splicing of nonapeptides excised from a pair of prepropeptide precursors. Thus, elucidation of the θ-defensin biosynthetic pathway provides an opportunity to identify novel factors involved in this unique process. We incorporated the θ-defensin precursor, proRTD1a, into a bait construct for a yeast two-hybrid screen that identified rhesus macaque stromal cell-derived factor 2-like protein 1 (SDF2L1), as an interactor. SDF2L1 is a component of the endoplasmic reticulum (ER) chaperone complex, which we found to also interact with α- and β-defensins. However, analysis of the SDF2L1 domain requirements for binding of representative α-, β-, and θ-defensins revealed that α- and β-defensins bind SDF2L1 similarly, but differently from the interactions that mediate binding of SDF2L1 to pro-θ-defensins. Thus, SDF2L1 is a factor involved in processing and/or sorting of all three defensin subfamilies.Mammalian defensins are tridisulfide-containing antimicrobial peptides that contribute to innate immunity in all species studied to date. Defensins are comprised of three structural subfamilies: the α-, β-, and θ-defensins (1). α- and β-Defensins are peptides of about 29–45-amino acid residues with similar three-dimensional structures. Despite their similar tertiary conformations, the disulfide motifs of α- and β-defensins differ. Expression of human α-defensins is tissue-specific. Four myeloid α-defensins (HNP1–4) are expressed predominantly by neutrophils and monocytes wherein they are packaged in granules, while two enteric α-defensins (HD-5 and HD-6) are expressed at high levels in Paneth cells of the small intestine. Myeloid α-defensins constitute about 5% of the protein mass of human neutrophils. HNPs are discharged into the phagosome during phagocytic ingestion of microbial particles. HD-5 and HD-6 are produced and stored as propeptides in Paneth cell granules and are processed extracellularly by intestinal trypsin (2). β-Defensins are produced primarily by various epithelia (e.g. skin, urogenital tract, airway) and are secreted by the producing cells in their mature forms. In contrast to pro-α-defensins, which contain a conserved prosegment of ∼40 amino acids, the prosegments in β-defensins vary in length and sequence. θ-Defensins are found only in Old World monkeys and orangutans and are the only known circular peptides in animals. These 18-residue macrocyclic peptides are formed by ligation of two nonamer sequences excised from two precursor polypeptides, which are truncated versions of ancestral α-defensins. Like myeloid α-defensins, θ-defensins are stored primarily in neutrophil and monocyte granules (3).Numerous laboratories have demonstrated that the antimicrobial properties of defensins derive from their ability to bind and disrupt target cell membranes (4), and studies have shown defensins to be active against Gram-positive and -negative bacteria (5), viruses (6–9), fungi (10, 11), and parasites such as Giardia lamblia (12). Defensins also play a regulatory role in acquired immunity as they are known to chemoattract T lymphocytes, monocytes, and immature dendritic cells (13, 14), act as adjuvants, stimulate B cell responses, and up-regulate proliferation and cytokine production by spleen cells and T helper cells (15, 16).Defensins are produced as pre-propeptides and undergo post-translational processing to form the mature peptides. While much has been learned about regulation of defensin expression, little is known about the factors involved in their biosynthesis. Valore and Ganz (17) investigated the processing of defensins in cultured cells and demonstrated that maturation of HNPs occurs through two proteolytic steps that lead to formation of mature α-defensins, but the proteases involved have yet to be identified. Moreover, there are virtually no published data regarding endoplasmic reticulum (ER)² factors that are responsible for the folding, processing, and sorting steps necessary for defensin maturation and secretion or trafficking to the proper subcellular compartment. It is likely that several chaperones, proteases, and protein-disulfide isomerase (PDI) family proteins are involved. Consistent with this possibility, Gruber et al. (18) recently demonstrated the role of a PDI in biosynthesis of cyclotides, small ∼30-residue macrocyclic peptides produced by plants.The primary structures of α- and θ-defensin precursors are closely related. We therefore undertook studies to identify proteins that interact with representative propeptides of each defensin subfamily with the goal of determining common and unique processes that regulate biosynthesis of α- and θ-defensins. We used two-hybrid analysis to first identify interactors of the θ-defensin precursor, proRTD1a. As described, we identified SDF2L1, a component of the ER-chaperone complex as an interactor, and showed that it also specifically interacts with α- and β-defensins. This suggests that SDF2L1 is involved in the maturation/trafficking of defensins at a step common to all three subfamilies of mammalian defensins.     

α<Subscript>1</Subscript>-Thymosin, α<Subscript>2</Subscript>-interferon,and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K _d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K _i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.     

β- Alanine,a Possible Neurotransmitter in the Visual System?

Abstract— The chemically evoked efflux of endogenous amino acids from perfused rabbit superior colliculus (SC) slices was studied. Amino acids in the perfusates were determined fluorimetrically with a precolumn derivatisation method and subsequent separation on an HPLC column. Potassium-induced depolarisation caused a calcium-dependent release of β-alanine, GABA, glutamate, and aspartate. Veratridine-evoked efflux was essentially similar in selectivity and could be blocked by tetrodotoxin (TTX). The results are indicative of a neurotransmitter role of β-alanine, GABA, glutamate, and aspartate in the SC of the rabbit.     

Meet the new mammoth,same as the old? Resurrecting the <Emphasis Type="Italic">Mammuthus primigenius</Emphasis>

Media reporters often announce that we are on the verge of bringing back the woolly mammoth, even while there is growing consensus among scientists that resurrecting the mammoth is unlikely. In fact, current “de-extinction” efforts are not designed to bring back a mammoth, but rather adaptations of the mammoth using close relatives. For example, Harvard scientists are working on creating an Asian elephant with the thick coat of a mammoth by merging mammoth and elephant DNA. But how should such creatures be classified? Are they elephants, mammoths, or both? Answering these questions requires getting clear about the concept of reproduction. What I hope to show is that with an appropriate notion of reproduction—one for which I will argue—resurrecting a member of Mammuthus primigenius is a genuine possibility.     

Object Depots in the Genus <Emphasis Type="Italic">Pogonomyrmex</Emphasis>: Exploring the “Who,” What,When, and Where

Harvester ants of the genus Pogonomyrmex collect and deposit many items on top of their nests. The depots of P. badius consist mostly of small charcoal fragments, while those of other species are primarily pebbles. Mature colonies can have hundreds of thousands of objects in their depot. In P. badius, the distributions of midden and charcoal about the mound are not completely overlapping, but are positively correlated in areas of overlap. Charcoal depots are isometric with colony size, but the amount of charcoal per colony size varies with season and site. The absence of the depot stimulates collection of nonfood objects. Recruitment to objects only occurs in the presence of food, and foragers choose objects based on size, but not color. An overview of current knowledge concerning depots in the genus indicates that depot formation is likely the ancestral state in the North American species. Furthermore, it is likely that selection is operating indirectly on these depots through selection on many dependent tasks.     

Is sexual segregation in the guppy, <Emphasis Type="Italic">Poecilia reticulata</Emphasis>, consistentwith the predation risk hypothesis?

We examined the importance of sex differences in predation risk in generating sexual segregation in the guppy, Poecilia reticulata. We hypothesised that sex differences in predation risk will result in habitat segregation and ultimately social segregation of the sexes, with the more vulnerable sex (males in this case) using safer habitats. In accordance with the predation risk hypothesis we observed sexual segregation in a population associated with high but not low predation risk. Under high predation risk we observed a larger proportion of males in shallow marginal habitats resulting in habitat segregation and ultimately social segregation of the sexes. Furthermore, habitat segregation by sex was associated with habitat segregation by body length with shoals in deeper water having a larger mean body length. Shoaling fish species have been key models in investigating group living, and further research directed towards understanding sexual segregation in other fish species would be valuable.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>, the Baker’s Yeast,suppresses the growth of Ehrlich carcinoma-bearing mice

This study was undertaken to evaluate the effectiveness and mechanisms of anti-tumor activity of Baker’s yeast, Saccharomyces cerevisiae, in immunocompetent mice. Swiss albino mice were inoculated intramuscularly in the right thigh with Ehrlich Ascites Carcinoma (EAC) cells. At day 8, mice bearing Solid Ehrlich Carcinoma tumor (SEC) were intratumorally (IT) injected with killed S. cerevisiae (10 × 10⁶ and 20 × 10⁶ cells) for 35 days. Histopathology of yeast-treated mice showed extensive tumor degeneration, apoptosis, and ischemic (coagulative) and liquefactive necrosis. These changes are associated with a tumor growth curve that demonstrates a significant antitumor response that peaked at 35 days. Yeast treatment (20 × 10⁶ cells) three times a week resulted in a significant decrease in tumor volume (TV) (67.1%, P < 0.01) as compared to PBS-treated mice. The effect was determined to be dependent on dose and frequency. Yeast administered three and two times per week induced significant decrease in TV as early as 9 and 25 days post-treatment, respectively. Administration of yeast significantly enhanced the recruitment of leukocytes, including macrophages, into the tumors and triggered apoptosis in SEC cells as determined by flow cytometry (78.6%, P < 0.01) at 20 × 10⁶ cells, as compared to PBS-treated mice (42.6%). In addition, yeast treatment elevated TNF-α and IFN-γ plasma levels and lowered the elevated IL-10 levels. No adverse side effects from the yeast treatment were observed, including feeding/drinking cycle and life activity patterns. Indeed, yeast-treated mice showed significant final body weight gain (+21.5%, P < 0.01) at day 35. These data may have clinical implications for the treatment of solid cancer with yeast, which is known to be safe for human consumption.     

Biology of the electric eel, <Emphasis Type="Italic">Electrophorus electricus</Emphasis>, Linnaeus, 1766 (Gymnotiformes: Gymnotidae) on the floodplain of the Curiaú River,eastern Amazonia

The evaluation of the growth patterns and reproductive strategies of fish species are vitally important for the understanding of their biology and the management of stocks. The present study focused on the Amazonian electric eel (Electrophorus electricus), which is capable of producing an electrical discharge of up to 800 V. Specimens were collected on a monthly schedule from a floodplain in the eastern Amazon basin. The gonads of these specimens were examined, and the sex ratio, growth parameters, population structure, body size at first gonadal maturation, and the gonadosomatic index were determined. A balanced sex ratio was found. Males were larger than females, and both sexes presented isometric growth, which is unusual in species with an elongated anatomy. This isometry may be related to the reduction of the coelomic cavity, and its position near the head. The spawning period coincided with the start of the rainy season, and continued until high water, with the variation in gonadal development following the fluctuations in precipitation and river water levels. The asymptotic body length in both sexes was relatively large, and was inversely related to the growth coefficient (k), with a slower growth rate being recorded in the males. Mortality rates were relatively low in comparison with most species of tropical fish. The larger size of the male may be related to their role in parental care, and sexual selection on the part of the females. These findings may be important for the management of wild stocks, as well as captive rearing.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> SERK1 protein interacts with the AAA-ATPase <Emphasis Type="Italic">At</Emphasis>CDC48, the 14-3-3 protein GF14λ and the PP2C phosphatase KAPP

Leucine-rich repeat (LRR)-containing transmembrane receptor-like kinases (RLKs) are important components of plant signal transduction. The Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 (AtSERK1) is an LRR-RLK proposed to participate in a signal transduction cascade involved in embryo development. By yeast two-hybrid screening we identified AtCDC48, a homologue of the mammalian AAA-ATPase p97 and GF14, a member of the Arabidopsis family of 14-3-3 proteins as AtSERK1 interactors. In vitro, the AtSERK1 kinase domain is able to transphosphorylate and bind both AtCDC48 and GF14. In yeast, AtCDC48 interacts with GF14 and with the PP2C phosphatase KAPP. In plant protoplasts AtSERK1 interacts with GF14.     

Analyzing the Interaction of RseA and RseB, the Two Negative Regulators of the ��E Envelope Stress Response, Using a Combined Bioinformatic and Experimental Strategy

The Escherichia coli envelope stress response is controlled by the alternative sigma factor, σ^E, and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects onσ^E activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disulfide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage.The Escherichia coli σ^E-mediated envelope stress response is the major pathway to ensure homeostasis in the envelope compartment of the cell (1-3). σ^E regulon members encode periplasmic chaperones and proteases, the machinery for inserting β-barrel proteins into the outer membrane and components controlling the synthesis and assembly of LPS (4-6). This pathway is highly conserved among γ-proteobacteria (6).The σ^E response is initiated when periplasmic protein folding and assembly is compromised (7-9). During steady state growth, σ^E is inhibited by its antisigma factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to σ^E with picomolar affinity (10-13). Accumulation of unassembled porin monomers serves as a signal to activate the DegS protease to cleave RseA in its periplasmic domain (14, 15). This initiates a proteolytic cascade in which RseP cleaves periplasmically truncated RseA near or within the cytoplasmic membrane to release the RseA_cytoplasmic-σ^E complex, and cytoplasmic ATP-dependent proteases complete the degradation of RseA thereby releasing active σ^E (16-19).RseB, a second negative regulator of the envelope stress response (11, 20, 21), binds to the periplasmic domain of RseA with nanomolar affinity. RseB is an important regulator of the response (2, 22, 23). It prevents RseP from degrading intact RseA, thereby ensuring that proteolysis is initiated only when the DegS protease is activated by a stress signal (21). Additionally, RseB prevents activated DegS from cleaving RseA, suggesting that interaction of RseB with RseA must be altered before the signal transduction cascade is activated (23).The goal of the present studies was to explore how RseB binds to RseA. The interaction partner of RseB is the unstructured periplasmic domain of RseA (RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major binding determinant to RseB (23, 24). This peptide alone binds RseB with 6 μm affinity, and deleting this region abrogates binding to RseB (23). Additional regions of RseA-peri also contribute to RseB binding, as intact RseA-peri binds with 20 nm affinity to RseB (23). Much less is known about the regions of RseB required for interaction with RseA. RseB is homodimeric two-domain protein, whose large N-terminal domain shares structural homology with LolA, a protein that transports lipoproteins to outer membrane (24, 25). The smaller C-terminal domain is connected to the N-terminal domain by a linker, and the two domains share a large interface, which may facilitate interdomain signaling. Glutaraldehyde cross-linking studies indicate that the C-terminal domain interacts with RseA, but the regions of interaction were not identified (25).In the present report, we study the interaction of RseB and RseA. We establish that both domains of RseB interact with RseA-peri. Using a global sequence alignment, we discovered several regions in RseA and RseB that had high sequence similarity, despite the low overall sequence similarity between these two proteins, a finding that was independently confirmed by a local sequence similarity algorithm. This suggested that these regions were functionally dependent, and we performed a set of mutagenesis experiments designed to test this idea. Our studies of the binding properties of these mutants revealed that regions in both the N terminus and C terminus of RseB modulate interaction with RseA. Moreover, genetic suppression analysis and cysteine-mediated disulfide bond formation suggest that the region of RseA/B with highest similarity (RseA residues 165-191 (major binding determinant in RseA) and RseB residues 233-258) are interacting partners.     

Theoretical study on the substitution reactions of the germylenoid H<Subscript>2</Subscript>GeLiF with SiH<Subscript>3</Subscript>X (X = F,Cl, Br)

The substitution reactions of H₂GeLiF (G) with SiH₃X (X = F, Cl, Br) were investigated using calculations performed at the QCISD/6-311++G (d, p)//B3LYP/6-311+G (d, p) level of theory. The results led to the following conclusions. (i) The substitutions are nucleophilic reactions. There are two substitution paths, I and II, which both lead to the germane H₂GeFSiH₃. The enantiomers of this germane are obtained via these two paths if an H in SiH₃X is replaced with a different group or atom. (ii) Both substitution pathways show the same order of barrier heights (SiH₃F > SiH₃Cl > SiH₃Br). The difference between the bond energies of Li–X and Si–X may explain the precedence among the substitution reactions of G with SiH₃X. Path I has a lower activation barrier than path II, indicating that path I is more favorable. (iii) Comparison between the relevant insertion and substitution reactions shows that substitutions are more favorable and that the substitution product H₂GeFSiH₃ predominates over the insertion product. (iv) The substitution reactions of H₂GeLiF with SiH₃X are exothermic.