Biosynthesis of natural-synthetic hybrid copolymers: polyhydroxyoctanoate-diethylene glycol

A new natural-synthetic hybrid biomaterial has been isolated from the growth of Pseudomonas oleovorans in the presence of diethylene glycol (DEG). DEG was consumed by P. oleovorans with 20 mM sodium octanoate in modified E* medium, but its presence in the fermentation medium retarded cell growth and viability, influencing production and composition of polyhydroxyalkanoates with medium chain length substituents (mclPHAs) and consequently attenuating PHA yield. DEG affected the composition of the mclPHA with an increase in the C8 component: polyhydroxyoctanoate (PHO). Gas chromatography-mass spectrometry (GC-MS) was used to quantitatively monitor DEG in the system and reveal its cellular adsorption and penetration. Intracellularly, the DEG significantly reduced the molar mass of the mclPHA; PHO with a bimodal distribution of high and low molecular weight fractions was observed. 1H NMR, 2-D COSY, and heteronuclear single quantum coherence spectra confirmed that the high molecular weight fraction consisted of PHO chains terminated by DEG. Thus, the synthesis of this natural-synthetic hybrid copolymer, PHO-DEG, opens the way for microbial synthesis of a wide variety of PHA-DEG copolymers with a range of bioactive properties.     

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.     

Tritrichomonas foetus: characterization of isolates and partial purification of a secreted cytotoxin

Putative virulence factors including extracellular proteases, hemagglutinin, hemolysins, and soluble cytotoxins may play significant roles in the pathogenesis of trichomoniasis. The cytotoxicity, hemagglutinating, and hemolytic activity of Tritrichomonas foetus isolate ATCC #30003 and several field isolates were compared. All isolates were hemolytic toward mouse and bovine erythrocytes but not other tested species. The isolates varied significantly in hemagglutinating ability and cytotoxin production. A 40,000 Da soluble cytotoxin was partially purified and characterized. Chromatography separated cytotoxic activity from hemagglutinating and hemolytic activity but not from protease activity. However, protease assays indicated that protease activity was inversely correlated with cytotoxic activity. Characterization studies indicated that cytotoxic activity was destroyed by heat and acidic conditions but repeated freeze/thawing did not diminish activity. Target cell specificity assays showed Henle cells were twice as sensitive to the effects of the cytotoxin as Vero cells. These results suggest that T. foetus isolates vary in the production of virulence factors and produce a soluble relatively stable non-protease cytotoxic protein capable of killing cultured mammalian cells in vitro.     

Novel secretory vesicle serpins,endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.     

A "dock, lock, and latch" structural model for a staphylococcal adhesin binding to fibrinogen

Gram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bbeta chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic "dock, lock, and latch" mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria.     

Influence of normal daytime fat deposition on laboratory measurements of torpor use in territorial versus nonterritorial hummingbirds

Fat deposition and torpor use in hummingbirds exhibiting distinct foraging styles should vary. We predicted that dominant territorial hummingbirds will use torpor less than subordinate nonterritorial species because unrestricted access to energy by territory owners allows for fat storage. Entry into torpor was monitored using open-flow respirometry on hummingbirds allowed to accumulate fat normally during the day. Fat accumulation was measured by solvent fat extraction. Territorial blue-throated hummingbirds (Lampornis clemenciae) had the highest fat accumulation and used torpor only 17% of the time. Fat storage by L. clemenciae averaged 26% of lean dry mass (LDM) in 1995 and 18% in 1996, similar to that measured for other nonmigratory birds. Fat storage by magnificent hummingbirds (Eugenes fulgens; trapliner) and black-chinned hummingbirds (Archilochus alexandri; nectar robber) averaged 19% and 16% of LDM, respectively, and they used torpor frequently (64% and 92% of the time, respectively). All species initiated torpor if total body fat dropped below 10% of LDM, indicating the existence of a torpor threshold. The ability of L. clemenciae to store enough fat to support nighttime metabolism is likely an important benefit of territoriality. Likewise, frequent torpor use by subordinates suggests that natural restrictions to energy intake can impact their energy budget, necessitating energy conservation by use of torpor.     

Expression and mutagenesis of the novel serpin endopin 2 demonstrates a requirement for cysteine-374 for dithiothreitol-sensitive inhibition of elastase

The primary sequence of the serpin endopin 2 predicts a reactive site loop (RSL) region that possesses high homology to bovine elastase inhibitor, suggesting inhibition of elastase. Moreover, endopin 2 possesses two cysteine residues that implicate roles for reduced Cys residue(s) for inhibitory activity. To test these predicted properties, mutagenesis and chemical modification of recombinant endopin 2 were performed to examine the influence of dithiothreitol (DTT), a reducing agent, on endopin 2 activity. Endopin 2 inhibited elastase in a DTT-dependent manner, with enhanced inhibition in the presence of DTT. The stoichiometry of inhibition in the presence of DTT occurred at a molar ratio of endopin 2 to elastase of 8/1, resulting in complete inhibition of elastase. However, a higher molar ratio (25/1) was required in the absence of DTT. DTT enhanced the formation of SDS-stable complexes of endopin 2 and elastase, a characteristic property of serpins. Site-directed mutagenesis of endopin 2, with substitution of Ala for Cys-232 or Cys-374, demonstrated that Cys-374 (but not Cys-232) was required for the DTT-sensitive nature of endopin 2. Chemical modification of Cys-374 by bis(maleimido)ethane also reduced inhibitory activity. Modified electrophoretic mobilities of mutant endopin 2 suggested the presence of intramolecular disulfide bonds; in addition, chemical modification suggested that Cys-374 influences the electrophoretic and conformational properties of endopin 2. Moreover, the reducing agent glutathione enhanced endopin 2 activity, suggesting that glutathione can function as an endogenous reducing agent for endopin 2 in vivo. These findings demonstrate the importance of Cys-374 for DTT-sensitive inhibition of elastase by endopin 2.     

Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A(332-364); identity of the peptide was confirmed by diagnostic Met(346) oxidation. Further analysis revealed two additional forms: bovine chromogranin A(333-364) and A(343-362). Synthetic longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC(50) approximately 2.01 versus approximately 0.35 microm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.     

Naturally Occurring Genetic Variants in Human Chromogranin A (CHGA) Associated with Hypertension as well as Hypertensive Renal Disease

Chromogranin A (CHGA) plays a fundamental role in the biogenesis of catecholamine secretory granules. Changes in storage and release of CHGA in clinical and experimental hypertension prompted us to study whether genetic variation at the CHGA locus might contribute to alterations in autonomic function, and hence hypertension and its target organ consequences such as hypertensive renal disease (nephrosclerosis). Systematic polymorphism discovery across the human CHGA locus revealed both common and unusual variants in both the open reading frame and such regulatory regions as the proximal promoter and 30-UTR. In chromaffin cell-transfected CHGA 30-UTR and promoter/luciferase reporter plasmids, the functional consequences of the regulatory/non-coding allelic variants were documented. Variants in both the proximal promoter and the 30-UTR displayed statistical associations with hypertension. Genetic variation in the proximal CHGA promoter predicted glomerular filtration rate in healthy twins. However, for hypertensive renal damage, both end-stage renal disease and rate of progression of earlier disease were best predicted by variants in the 30-UTR. Finally, mechanistic studies were undertaken initiated by the clue that CHGA promoter variation predicted circulating endothelin-1. In cultured endothelial cells, CHGA triggered co-release of not only the vasoconstrictor and pro-fibrotic endothelin-1, but also the pro-coagulant von Willebrand Factor and the pro-angiogenic angiopoietin-2. These findings, coupled with stimulation of endothelin-1 release from glomerular capillary endothelial cells by CHGA, suggest a plausible mechanism whereby genetic variation at the CHGA locus eventuates in alterations in human renal function. These results document the consequences of genetic variation at the CHGA locus for cardiorenal disease and suggest mechanisms whereby such variation achieves functional effects.     

Mannosylated saponins based on oleanolic and glycyrrhizic acids. Towards synthetic colloidal antigen delivery systems

Immunostimulatory saponin based colloidal antigen delivery systems show promise as adjuvants for subunit vaccines. For this reason, allyl oleanolate was glycosylated at the 3-position using trichloroacetimidate donors to give monodesmodic saponins following deprotection. Bisdesmodic saponins were synthesized by double glycosylation at the 3- and 28-positions of oleanolic acid. When formulated together with cholesterol and phospholipids, ring-like, helical and rod-like nanostructures were formed depending on the saponin concentrations used. As an indication of adjuvant activity, the ability of these formulations, and the saponins by themselves, to induce dendritic cell maturation was measured, but no significant activity was observed.