Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7-501 using ion-exchange chromatography,hydrophobic interaction chromatography,and Rotofor® isoelectric focusing

Hyalophora cecropia pupae were infected by Enterobacter cloacae C7-501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non-specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7-501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.     

Phylogeny of Selected Maxillopodan and Other Crustacean Taxa Based on 18S Ribosomal Nucleotide Sequences: A Preliminary Analysis

Relationships among the following taxa were examined based on 18S ribosomal RNA and DNA nucleotide sequences: Branchiopoda (Branchinecta packardi), Ostracoda (two podocopid species), Branchiura (Argulus nobilis), Pentastomida (Porocephalus crotali), Copepoda (Calanus pacificus, Macrocyclops albidus), Thoracica (Balanus eburneus, Calantica villosca), Acrothoracica (Trypetesa lampas), and Decapoda (Procambarus leonensis, Callinectes sapidus). Phylogenetic relationships were inferred by maximum parsimony and results evaluated by bootstrapping. With Branchinecta packardi as an outgroup, the following tree was inferred: the first branch leads to a node that joins a pentastome, a branchiuran and the two ostracodes; the second branch leads to a node that has the two copepods as a sister taxon to a clade that includes the acrothoracican and thoracicans; the last branch leads to a node joining the two decapods. The analyses suggest: (1) acrothoracicans diverged very early from the cirripede line and are not derived from a lepadomorph-like ancestor; (2) branchiurans are not related to either copepods or thecostracans but are closely allied with pentastomes; and (3) the Maxillopoda, broadly defined, is not a monophyletic taxon. These results differ from previous studies of these groups.     

EVOLUTIONARY IMPLICATIONS OF INTRASPECIFIC CHLOROPLAST DNA VARIATION IN DWARF DANDELIONS (KRIGIA; ASTERACEAE)

Intraspecific chloroplast DNA polymorphisms were examined for 51 populations of seven species in the genus Krigia. A total of 1,100 restriction sites was surveyed and 46 of these were variable at the intraspecific level. Twenty-two of the variable sites were found within K. virginica, giving this species one of the highest levels of intraspecific chloroplast DNA divergence of any examined species. In contrast, no restriction site variation was detected within K. dandelion, K. wrightii, and K. occidentalis. Five polymorphisms were identified from the 16 populations of the K. cespitosa-gracilis complex, but no mutations distinguished the K. cespitosa and K. gracilis types. Krigia montana and K. biflora showed 11 and eight restriction site polymorphisms, respectively. The chloroplast genome of the hexaploid K. montana was derived from the diploid K. biflora rather than the tetraploid K. montana. High levels of polymorphism were found in species having different ploidy levels, such as K. virginica, K. biflora, and K. montana. Furthermore, most mutations found in these three species were recorded from the tetraploid lineages. As a result, evolutionary rates between different ploidy levels differ significantly. The chloroplast DNA restriction site data suggest that all surveyed populations of the autotetraploid K. virginica originated from a common ancestor. Our results also indicate that certain regions of the chloroplast genome have changed more rapidly than others and have the potential to resolve evolutionary questions at the population level.     

REGENERATION AND SEXUAL DIFFERENTIATION OF GRIFFITHSIA JAPONICA (CERAMIACEAE,RHODOPHYTA) THROUGH SOMATIC CELL FUSION1

Hybrid cells were obtained from somatic cell fusion among male, female, and tetrasporangial plants in Griffithsia japonica Okamura by a wound-healing process. Isolated fusion cells regenerated new mature plants with mixed reproductive structures. The plants regenerated from hybrid cells between male and female plants developed into 1) spermatangiate, 2) carpogonial, 3) bisexual with spermatangia and carpogonial branches, 4) mixed-phase with spermatangia and tetrasporangia, or 5) bisexual/mixed-phase plants with spermatangia, carpogonial branches, and tetrasporangia. About 70% of the plants regenerated from hybrid cells between male and female plants produced tetrasporangia that were always formed with spermatangia on a single cell. Some of those tetrasporangia released tetraspores, six of which gave rise to mature plants. The plants regenerated from hybrid cells between male and tetrasporangial plants developed into spermatangiate, tetrasporangiate, or mixed-phase plants with spermatangia and tetrasporangia. The plants regenerated from hybrid cells between female and tetrasporangial plants developed into carpogonial, tetrasporangiate, or mixed-phase plants with carpogonial branches and tetrasporangia. All types of reproductive structures we re functional.     

Toxoplasma antibody titers by indirect latex agglutination test in patients of Kangnam St. Mary's Hospital and Cheju Medical Center

Total 2,829 persons consisted of 1,019 general patients and 1,030 asthma-suspected patients who visited Kangnam St. Mary's Hospital and 780 general patients who visited Cheju Medical Center were examined for the antibody titers of Toxoplasma by indirect latex agglutination (ILA) test. Nineteen out of 1,019(1.86%) cases in general patients group, 11 out of 1,030(1.07%) cases in asthma patients group, and 45 out of 780(5.77%) cases in Cheju patients group showed positive ILA titers. Concerned with the age and ILA positive cases, general and asthma patients expressed more cases at thirties to sixties while Cheju patients showed high incidence at children and adolescents in addition to the above mentioned ages. Frequencies of ILA positive titers were highest in 1:32 and 1:64, and some cases showed 1:2,048 or higher titers.     

Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin. Lack of evidence for unique oligosaccharide sequences mediating binding

Previous studies have shown that the mucin-type polypeptidesGlyCAM-1, CD34, and MAdCAM-1 can function as ligands for L-selectinonly when they are synthesized by the specialized high-endothelialvenules (HEV) of lymph nodes. Since sialylation, sulfation,and possibly fucosylation are required for generating recognition,we reasoned that other mucins known to have such componentsmight also bind L-selectin. We show here that soluble mucinssecreted by human colon carcinoma cells, as well as those derivedfrom human bronchial mucus can bind to human L-selectin in acalcium-dependent manner. As with GlyCAM-1 synthesized by lymphnode HEY, 2–3 linked sialic acids and sulfation seem toplay a critical role in generating this L-selectin binding.In each case, only a subset of the mucin molecules is recognizedby L-selectin. Binding is not destroyed by boiling, suggestingthat recognition may be based primarily upon carbohydrate structures.Despite this, O-linked oligosaccharide chains released fromthese ligands by beta-elimination do not show any detectablebinding to L-selectin. Following protease treatment of the ligands,binding persists in a subset of the resulting fragments, indicatingthat specific recognition is determined by certain regions ofthe original mucins. How ever, O-linked oligosaccharides releasedfrom the subset of non-binding mucin fragments do not show verydifferent size and charge profiles compared to those that dobind. Furthermore, studies with polylactosamine-degrading endoglycosidasessuggest that the core structures involved in generating bindingcan vary among the different ligands. Taken together, thesedata indicate that a single unique oligosaccharide structuremay not be responsible for high-affinity binding. Rather, diversemucins with sialylated, sulfated, fucosylated lactosamine-typeO-linked oligosaccharides can generate high-affinity L-selectinligands, but only when they present these chains in unique spacingand/or clustered combinations, presumably dictated by the polypeptidebackbone. L-selectin mucins sialic sialic acid sulfate adhesion     

Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium

Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σ^s-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli . The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.     

Photo-induced activation of cytochrome P450/reductase fusion enzyme coupled with spinach chloroplasts

Summary Photoactivation of cytochrome P450 monooxygenase was studied using a combination of spinach chloroplasts and yeast microsomes containing rat P4501A1/yeast reductase fusion enzyme. Under illumination, in the reaction mixture, NADP was reduced, transferring electrons to the P450/reductase fusion enzyme to convert 7-ethoxycoumarin to 7-hydroxycoumarin.     

Mechancial properties of bat wing membrane skin

The skin of the bat wing in functionally unique among mammals: it serves as a major locomotor organ in addition to its protective and regulatory functions. We used tensile testing to investigate the mechanical capabilities of wing membrane skin, and compared stiffness, strength, load at failure, and energy absorption among specific wing regions and among a variety of bat taxa. We related these characteristics to the highly architectural fibrous supporting network of the wing membrane. We found that all material properties showed a strong anisotropy. In particular, wing membrane skin shows maximum stiffness and stregth parallel to the wing skeleton, and greatest extensibility parallel to the wing's trailing edge. We also found significant variation among wing regions. The uropatagium (tail membrane) supported the greatest load at failure, and the plagiopatagium (proximal wing membrane between laterl body wall and hand skeleton) is the weakest and most extensible part of the wing. We believe that the increased load bearing ability of the uropatagium relats to its key role in capture of insect prey, and that the great extensibility of the plagiopatagium promotes development of camber near the wing's centre of lift. In interspecific comparisons, energy absorpion and load to failure were greatest in Artibeus jamaicensis , the largest bat in our sample and the species with the highest wing loading, suggesting that wing loading may play a role in dictating the fuctional design of wing membranes.