High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

Efficacy of methyl 5(6)-(alpha-hydroxyphenylmethyl) benzimidazole-2-carbamate, a metabolite of mebendazole, against developing forms of experimental helminth parasites.

Methyl 5(6)-(alpha-hydroxyphenylmethyl) benzimidazole-2- carbamate, a metabolite of mebendazole, was evaluated against metamorphic forms of Ancylostoma ceylanicum in hamsters, Nippostrongylus brasiliensis in rats and cysticercoids of Hymenolepis nana in grain beetles. The test compound offered better action than mebendazole except against H. nana cysticercoids where the activity of the compound and mebendazole was comparable, but was inferior to the standard cestodicidal drug, praziquantel. The results suggest that the action was better by ip route compared to per os route of drug administration.     

Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin

The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbants in order to assess the magnitude of the interactions within the B-subunits. For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbants, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion. At pH 7.5 and in the absence of any additional perturbants, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacity function centered at 77 degrees C and characterized by a delta Hcal of 328 kcal/mol of B-subunit pentamer and delta Hvh/delta Hcal of 0.3. Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van't Hoff enthalpy. The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3. Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 degrees C. Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer. Similar behavior is obtained with GuHCl. In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes

Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50 degrees C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability under low ionic conditions, and the binding of spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluorescence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than that observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably increased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. In spite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.     

New erythrina alkaloids of Cocculus laurifolia

Four abnormal Erythrina alkaloids have been isolated from the leaves of Cocculus laurifolia. Of these, two new bases, isococculidine and coccoline, have been assigned the structures (3S,5S)-3,15-dimethoxyerythrin-1-ene and (3R,5S)-15-hydroxy-3-methoxyerythrin-1-en-8-one respectively.     

The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.     

Streptococcus pneumoniae hyaluronate lyase contains two non-cooperative independent folding/unfolding structural domains: characterization of functional domain and inhibitors of enzyme

Hyaluronate lyase contributes directly to bacterial invasion by degrading hyaluronan, the major component of host extracellular matrix of connective tissues. Streptococcus pneumoniae hyaluronate lyase (SpnHL) is built from two structural domains that interact through interface residues, in addition to being connected by a peptide linker. For the first time we demonstrate that the N- and C-terminal domains of SpnHL fold/unfold independent of each other suggesting the absence of any significant cooperative interactions between them. The C-terminal domain of SpnHL is less stable than the N-terminal domain against thermal and guanidine hydrochloride denaturation. The intact N-terminal domain was purified after limited proteolysis of SpnHL under conditions where only the C-terminal domain was unfolded. Isolated N-terminal domain of SpnHL had similar thermal stability as when present in the native enzyme and was found to be enzymatically active demonstrating that it is capable of carrying out enzymatic reaction on its own. Functional studies demonstrated that guanidine hydrochloride, guanidine isothiocyanate, l-arginine methyl ester, and l-arginine inhibit the enzymatic activity of SpnHL at very low concentrations. This provides a lead for new chemical entities that can be exploited for designing effective inhibitors of SpnHL.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Insect feeding deterrent and growth inhibitory activities of scopoletin isolated from Artemisia annua against Spilarctia obliqua (Lepidoptera: Noctuidae)

Abstract Artemisia annua (Asteraceae) is well known for its antimalarial activities due to presence of the compound artemisinin. We isolated a methoxy coumarin from the stem part of A. annua and confirmed its identity as scopoletin through mass spectral data. The structure was established from ¹H‐nuclear magnetic resonance (NMR), ¹³C‐NMR. The compound scopoletin was evaluated for its feeding deterrence and growth inhibitory potential against a noxious lepidopteran insect, Spilartctia obliqua Walker. Scopoletin gave FD₅₀ (feeding deterrence of 50%) value of 96.7 μg/g diet when mixed into artificial diet. S. obliqua larvae (12‐day‐old) exposed to the highest concentration (250 μg/g diet) of scopoletin showed 77.1% feeding‐deterrence. In a growth inhibitory assay, scopoletin provided 116.9% growth inhibition at the highest dose of 250 μg/g diet with a GI₅₀ (growth inhibition of 50%) value of 20.9 μg/g diet. Statistical analysis showed a concentration‐dependent dose response relationship toward both feeding deterrent and growth inhibitory activities. Artemisinin is found mainly in the leaves of A. annua and not in the stems, which are typically discarded as waste. Therefore identification of scopoletin in stems of A. annua may be important as a source of this material for pest control.