Paleobiota from the Deccan volcano-sedimentary sequences of India: paleoenvironments,age and paleobiogeographic implications

Paleobiotic assemblages from the Deccan infra- and intertrappean beds are reviewed in great detail. Three distinct paleoenvironments (fluvio-lacustrine/terrestrial, brackish water and marine) have been identified within the infra- and intertrappean biotic assemblages of peninsular India. Recently, marine incursions have been recorded in a few of the Deccan intertrappean beds exposed in central and south-eastern India. The intertrappean beds have yielded marine planktic foraminiferans and freshwater/brackish water ostracods. The affinities of the paleobiotas are commonly considered to show a mixed pattern resulting from the addition of Gondwanan and Laurasian elements to endemic Indian taxa. During the last four decades, various biogeographic models (southern and northern connections) have been proposed to explain the presence of anomalous biogeographic biota in the Late Cretaceous of India. Based on the recovered fauna and flora assemblages, the Cretaceous–Paleogene boundary has been marked and a Late Cretaceous to Early Paleocene age has been assigned to these Deccan volcano-sedimentary sequences.     

A scaleable manufacturing process for pro-EP-B2, a cysteine protease from barley indicated for celiac sprue

Celiac Sprue is an inflammatory disease of the small intestine triggered by ingestion of dietary gluten, a family of glutamine and proline rich proteins found in common foodgrains such as wheat, rye, and barley. One potential therapy for this lifelong disease anticipates using an oral protease to detoxify gluten in vivo. Recent studies have shown that EP-B2 (endoprotease B, isoform 2) from barley is a promising example of such a glutenase, thus warranting its large-scale production for animal safety and human clinical studies. Here we describe a scaleable fermentation, refolding and purification process for the production of gram to kilogram quantities of pro-EP-B2 (zymogen form of EP-B2) in a lyophilized form. A fed-batch E. coli fermentation system was developed that yields 0.3-0.5 g purified recombinant protein per liter culture volume. Intracellular degradation of pro-EP-B2 during the fermentation was overcome by manipulating the fermentation temperature and duration of protein expression. A simple refolding protocol was developed using a fast dilution method to refold the enzyme at concentrations greater than 0.5 mg/mL. Kinetic analysis showed that pro-EP-B2 refolding is a first-order reaction with an estimated rate constant of 0.15 h(-1). A lyophilization procedure was developed that yielded protein with 85% recoverable activity after 7 weeks of storage at room temperature. The process was successfully scaled up to 100 L with comparable purity and recovery.     

Transglutaminase 2 undergoes a large conformational change upon activation

Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-Å resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.     

The loading module of rifamycin synthetase is an adenylation-thiolation didomain with substrate tolerance for substituted benzoates

The rifamycin synthetase is primed with a 3-amino-5-hydroxybenzoate starter unit by a loading module that contains domains homologous to the adenylation and thiolation domains of nonribosomal peptide synthetases. Adenylation and thiolation activities of the loading module were reconstituted in vitro and shown to be independent of coenzyme A, countering literature proposals that the loading module is a coenzyme A ligase. Kinetic parameters for covalent arylation of the loading module were measured directly for the unnatural substrates benzoate and 3-hydroxybenzoate. This analysis was extended through competition experiments to determine the relative rates of incorporation of a series of substituted benzoates. Our results show that the loading module can accept a variety of substituted benzoates, although it exhibits a preference for the 3-, 5-, and 3,5-disubstituted benzoates that most closely resemble its biological substrate. The considerable substrate tolerance of the loading module of rifamycin synthetase suggests that the module has potential as a tool for generating substituted derivatives of natural products.     

An antibiotic factory caught in action

The synthesis of aromatic polyketides, such as actinorhodin, tetracycline and doxorubicin, begins with the formation of a polyketide chain. In type II polyketide synthases (PKSs), chains are polymerized by the heterodimeric ketosynthase-chain length factor (KS-CLF). Here we present the 2.0-A structure of the actinorhodin KS-CLF, which shows polyketides being elongated inside an amphipathic tunnel approximately 17 A in length at the heterodimer interface. The structure resolves many of the questions about the roles of KS and CLF. Although CLF regulates chain length, it does not have an active site; KS must catalyze both chain initiation and elongation. We provide evidence that the first cyclization of the polyketide occurs within the KS-CLF tunnel. The mechanistic details of this central PKS polymerase could guide biosynthetic chemists in designing new pharmaceuticals and polymers.     

Heparin affects signaling pathways stimulated by fibroblast growth factor-1 and -2 in type II cells

Undersulfation of the basement membrane matrix of alveolar type II (AT2) cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these pneumocytes. Heparin, a model for sulfated components of basement membrane matrices, is known to inhibit fibroblast growth factor (FGF)-2-stimulated DNA synthesis as well as gene expression of FGF-2 and its receptor in AT2 cells. To determine whether these end points result from specific effects of heparin on FGF-related signaling pathways, isolated rat AT2 cells were treated with 100 ng/ml FGF-1 or FGF-2 in the presence of up to 500 microg/ml heparin. In addition, experiments were done on cells grown in the presence of 20 mM sodium chlorate (sulfation inhibitor). High-dose heparin reduced FGF-1- or FGF-2-stimulated phosphorylation of mitogen-activated protein kinase kinases (MEK1/2), p44/42 mitogen-activated protein kinases (MAPK/ERK1/2), stress-activated protein kinase/c-Jun NH(2)-terminal kinase, Akt/protein kinase B, and p90(RSK). FGF-2-stimulated signaling was more sensitive to heparin's effects than was signaling stimulated by FGF-1. Heparin had an additive effect on the reduced [(3)H]thymidine incorporation in FGF-2-treated AT2 cells caused by inhibition of the MEK/ERK pathway by the MEK inhibitor PD-98059. The data suggest that heparin's known capacity to alter DNA synthesis and, possibly, other biological end points is realized via cross talk between multiple signaling pathways.     

Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases

Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction.     

Regulation of osteoclastogenesis and RANK expression by TGF-beta1

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta1 (0.01-5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-beta1 resulted in a significant increase in the percentage of RANK+ RAW cells (P < 0.05), as well as an increase in the fluorescence intensity per cell (P < 0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-beta directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression.     

Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone

Parathyroid hormone (PTH) has biphasic effects on bone: continuous treatment is catabolic whereas intermittent treatment is anabolic. The mechanism(s) responsible for these differing effects are still unclear, partly because of the previous non-availability of a model system in which effects on both formation and resorption indices could be studied concomitantly. In cultured marrow cells from 6-week old C57BL/6 mice, we demonstrated that 4 days of intermittent PTH treatment increased mRNA for osteoblast differentiation markers (Runx2, alkaline phosphatase (AP), and type I procollagen (COL1A1) whereas continuous treatment resulted in production of large numbers of TRAP-positive multinucleated osteoclasts. Although IGF-I mRNA did not increase after intermittent treatment, it was consistently higher than after continuous treatment, and the addition of an anti-IGF-I neutralizing antibody prevented the increase in bone formation indices observed with intermittent treatment. By contrast, after continuous treatment, gene expression of RANK ligand (RANKL) was increased and that of osteoprotegerin (OPG) was decreased, resulting in a 25-fold increase in the RANKL/OPG ratio. In this model system, the data suggest that intermittent PTH treatment enhances osteoblast differentiation through an IGF-I dependent mechanism and continuous PTH treatment enhances osteoclastogenesis through reciprocal increases in RANKL and decreases in OPG.