Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.     

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

S100 alpha, CAPL, and CACY: molecular cloning and expression analysis of three calcium-binding proteins from human heart.

Elevated levels of intracellular calcium are a major cause of myocardial dysfunction. To find possible mediators of the deregulated calcium we searched for EF-hand calcium-binding proteins of the S100 family. By PCR technology we identified three members of the S100 protein family (S100 alpha, CACY, and CAPL) in the human heart. We cloned the corresponding cDNAs and examined their expression levels in various human tissues by Northern blot analysis. All three proteins are expressed at high levels in the human heart. Whereas CACY and CAPL mRNAs are expressed ubiquitously, S100 alpha mRNA is restricted to heart, skeletal muscle, and brain. Interestingly, the expression pattern of S100 alpha, CACY, and CAPL in human tissues differs significantly from that in rodent tissues.     

Volatile profiles of human skin cell cultures in different degrees of senescence

It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.     

Effect of separator and inoculum type on electricity generation and microbial community in single-chamber microbial fuel cells

Single-chamber microbial fuel cell (SMFC)-I consisted of 4 separator-electrode assemblies (SEAs) with two types of cation exchange membrane (CEM: Nafion and CMI 7000) and an anion exchange membrane (AEM: AMI 7001). SMFC-II consisted of 4 SEAs with Nafion and three types of nonwoven fabric. SMFC-I and -II were inoculated with anaerobic digested and activated sludge, respectively, and operated under fed-batch mode. In SMFC I, AEM-SEA showed a maximum power density (PD_max). Nafion-SEA showed a PD_max in SMFC II, which was similar to that of Nafion–SEA of SMFC I. Although different bacteria were developed in SMFC-I (Deltaproteobacteria and Firmicutes) and SMFC-II (Gammaproteobacteria, Betaproteobacteria and Bacteroidetes), the inoculum type little affects electricity generation. Variations of pH and oxygen in biofilm have influenced microbial community structure and electricity generation according to the electrode and separator material. Although the electricity generation of non-woven fabric-SEA was less than that of Nafion-SEA, the use of non-woven fabrics is expected to reduce the construction and operating costs of MFCs.     

Preference and performance of Lepidoptera varies with tree age in juniper woodlands

1. As trees age, they undergo significant physiological and morphological changes. Nevertheless, tree ontogeny and its impacts on herbivores are often overlooked as determinants of plant–herbivore population dynamics and the strength of plant–herbivore interactions. 2. Juniperus (Cupressaceae) is a dominant, long‐lived conifer that serves as the sole host to a specialised assemblage of caterpillars. Over the past 150 years, several juniper species in western North America have expanded their geographic occupancy at local and regional scales, which has resulted in an increase in the number of immature trees on the landscape. Using assays in the laboratory, the effects of tree ontogeny on caterpillar performance and oviposition preference for two juniper specialist caterpillars, Callophrys gryneus (Lycaenidae) and Glena quinquelinearia (Geometridae), were examined. The study considered whether responses to tree ontogeny were consistent across caterpillar species and juniper host species. 3. Tree age was found to be a reliable predictor of caterpillar performance, with caterpillars developing more quickly and growing larger when fed foliage from young trees. Differences in the phytochemical diversity between foliage from trees of different ages might help to explain observed differences in caterpillar performance. Interestingly, the specialist butterfly, C. gryneus, displayed an oviposition preference for foliage from old‐growth Juniperus osteosperma trees, despite the fact that larvae of this species performed poorly on older trees. 4. It is concluded that young juniper trees are an important resource for the specialised Lepidopteran community and that tree ontogeny is an important component of intraspecific variation, which contributes to the structure of plant–herbivore communities.     

The subunit composition of Portunus trituberculatus hemocyanin polymers

The subunit composition of isolated polymeric forms of Portunus trituberculatus hemocyanin were analysed by immunological techniques. The dodecamers contain four monomeric subunits corresponding to subunits I, II, III and IV, whereas the hexamers are devoid of subunit IV. These results suggest that subunit IV is required as a joining piece for the assembly of dodecamers.