Molecular characterisation of a novel thermophilic nitrile hydratase

The thermophilic soil isolate, Bacillus pallidus Dac521, expresses a constitutive nitrile hydratase. The purified enzyme was found to be a 110 kDa tetramer composed of two alpha and two beta subunits with molecular masses of 27 kDa and 29 kDa, respectively. The enzyme electrophoresed as a single protein band on native PAGE but two protein bands with isoelectric points of 4.7 and 5.5 on isoelectric focusing suggested the presence of isozymes. The purified enzyme was moderately thermostable up to 55 degrees C and the enzyme activity was stable over a broad pH range. Comparisons of the N-terminal amino acid sequences of the nitrile hydratase subunits with those of other nitrile hydratases showed up to 90% identity for the beta subunit sequence but no significant identity for the alpha subunit. The enzyme hydrolysed a narrow range of aliphatic substrates and did not hydrolyse any of the cyclic, hydroxy-, di- or aromatic nitriles tested. The activity was irreversibly inhibited by the aromatic nitrile, benzonitrile. The kinetic constants for acetonitrile, acrylonitrile and propionitrile compared favourably with those of mesophilic nitrile hydratases.     

The Norway rat as a reservoir host of Cryptosporidium parvum

The potential of Norway rats (Rattus norvegicus) to spread the parasite Cryptosporidium parvum was investigated by examining parasite prevalence in relation to the structure and movements of three permanent rat populations living on farmland in Warwickshire (UK) from October 1994 to March 1997. One population lived among a group of farm buildings housing cattle, while the other two had no contact with livestock, one living around a pond and its outflowing stream and the other on a rubbish tip. Overall, parasite occurrence was 24% (n = 438), but it varied according to body weight (age) with 40% of juveniles (< or =100 g) infected decreasing to 12% for adults >400 g, suggesting that actively breeding populations are potentially more likely to spread the parasite than non-breeding populations. There was no difference in prevalence between the three populations. The parasite was detected in more males (29%) than females (19%). Seasonally, on the livestock farm, prevalence was significantly lower in autumn (10%), but varied little (31-36%) from winter to summer. In contrast, on the arable farm, prevalence peaked in summer (50%) with a trough in winter (6%). Infection in rats appeared to last <67 days. Rats living on the livestock farm had home ranges largely confined to the cattle sheds, thereby maintaining a potential source of infection for livestock if rodent control was not part of a decontamination program. Equally, rats living around the pond on the arable farm provided a source of oocysts to contaminate the pond water, as well as being able to carry the parasite to nearby farm buildings or even to neighboring farms.     

The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein

Intermedilysin is a pore-forming cytolysin belonging to the streptolysin O gene family known as the 'Cholesterol-binding/dependent cytolysins' and is unique within the family in that it is highly humanspecific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome-embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome-binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human-specific step of intermedilysin action, that the host cell membrane-binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human-specific action step and the cell-binding site of domain 4 lies within the 56 amino acids of the C-terminal, excluding the undecapeptide region.     

Mechanisms of murine cranial suture patency mediated by a dominant negative transforming growth factor-beta receptor adenovirus

Using a physiologic model of mouse cranial suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the suture complex of the fusing posterior frontal compared with the patent sagittal suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior frontal suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the suture mesenchyme and subjacent dura mater during the early period of predicted posterior frontal suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.     

Thermotoga maritima IscU. Structural characterization and dynamics of a new class of metallochaperone

Members of the IscU family of proteins are among the most conserved of all protein groups, extending across all three kingdoms of life. IscU serves as a scaffold for the assembly of intermediate iron-sulfur cluster centers and further mediates delivery to apo protein targets. Several proteins that mediate delivery of single metal ions to apo targets (termed metallochaperones) have recently been characterized structurally. Each displays a ferredoxin-like betaalphabetabetaalphabeta motif as a structural core. Assembly and delivery of a polynuclear iron-sulfur cluster is, however, a more complex pathway and presumably would demand a distinctive protein mediator. Here, we demonstrate Thermotoga maritima IscU (Tm IscU) to display unique structural and motional characteristics that distinguish it from other members of this class of proteins. In particular, IscU adopts a mobile, physiologically relevant, molten globule-like state that is vastly different from the previously identified ferredoxin-like fold that has thus far been characterized for other metallochaperones. The secondary structural content of Tm IscU is consistent with previous circular dichroism measurements on apo and holo protein, consisting of six alpha-helices and three beta-strands, the latter forming an anti-parallel beta-sheet. Extensive dynamics studies are consistent with a protein that has reasonably well defined secondary structural elements, but with a tertiary structure that is fluxional among widely different conformational arrangements. Analogous conformational flexibility does not exist in other structurally characterized metallochaperones; however, such a dynamic molecule may account for the lack of long-range NOEs, and allow both for the flexibility that is necessary for the multiple roles of Fe-S cluster assembly, and recognition and delivery of that cluster to a target protein. Additionally, the fluxionality of IscU is unique in that the protein appears to be more compact (based on 1H/2H exchange, R1, R2, and NOE data) but yet more fluid (lack of long-range NOEs) than typical molten globule proteins.     

Characterization of the soluble domain of the ABC7 type transporter Atm1

Atm1 is an ABC transporter that is located in yeast mitochondria and has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins. The soluble nucleotide binding domain of Atm1 (Atm1-C) has been overexpressed in Escherichia coli, purified, and characterized. Dissociation constants (KD) for Atm1-C binding of ATP (KD approximately 97 microm, pH 7.3, and approximately 102 microm, pH 10.0) and ADP (KD approximately 43 microm, pH 7.3, and 92 microm, pH 10.0) were measured by fluorimetry. The higher binding affinity for ADP suggests that the transmembrane-spanning domain may be required to promote a structural change in the nucleotide binding domain to facilitate substrate export and ADP release. ADP also had an inhibitory effect on Atm1-C with an IC50 of 10 mm. The Michaelis-Menten constants Vmax, KM, and kcat of Atm1-C were measured as 1.822 microm min(-1), 513 microm, and 0.055 min(-1), respectively. The metal dependence of Atm1-C ATPase demonstrated a reactivity order of Mn2+ > Mg2+ > Co2+, while Mg2+ and Co2+ were both found to be inhibitory at higher concentrations. The pH profile and structural comparison with HisP are consistent with a role for His and Lys in promoting the ATPase activity. Structural analysis of Atm1-C by CD spectroscopy suggested a similarity of secondary structure to that found for a prokaryotic homologue (HisP), whereas modeling of the Atm1-C tertiary structure using HisP as a template is also consistent with a similarity in tertiary structure. Atm1-C tends to form a dimer or higher aggregation state at higher concentration; however, the concentration dependence of Atm1-C on ATPase activity and the results of a Hill analysis (napp = 1.1) demonstrated that there was essentially no cooperativity in ATP hydrolysis, in contrast to observations for the prokaryotic HisP transporter, which demonstrated full cooperativity for both full-length and the soluble domains. Accordingly, any cooperative response must be mediated through the transmembrane domain in the case of the eukaryotic Atm1 transporter.     

Plant hormone homeostasis and the control of avocado fruit size

Control of plant hormone homeostasis is crucial for normal organdevelopment in plants. To elucidate the contribution of plant hormonehomeostasis to fruit growth, tissue distribution and activity of xanthinedehydrogenase (XDH), abscisic aldehyde (AB-ald)- and indole acetaldehyde(IA-ald) oxidase, and cytokinin oxidase (CKOX) were determined in seed, seedcoat and mesocarp of normal 'Hass avocado and its small-fruitphenotype during the linear phase of growth. Activity of these enzymes wasrelated to the tissue content of indole-3-acetic acid (IAA) and abscisic acid(ABA). IA-ald oxidase was present only in seed tissue whereas AB-ald oxidase andXDH activity was found in seed and mesocarp tissue. Seed of the small'Hass fruit had increased XDH and AB-ald oxidase activity and highendogenous ABA, but reduced IA-ald oxidase activity and adenine. There was nodifference in seed, seed coat and mesocarp CKOX activity between normal andsmall fruit. Inhibition of XDH activity in whole fruit by treatment withallopurinol decreased IAA and increased ABA of seed tissue. In mesocarp ofripening fruit allopurinol increased ABA and IAA but had no effect on levels ofiP. Results indicate that activity of IA-ald and AB-ald oxidases in avocadofruit contribute to maintenance of the IAA/ABA ratio in seed and mesocarp tissueand that increased AB-ald oxidase, or reduced IA-ald oxidase, may be part of thesyndrome associated with the appearance of a small-fruit phenotype.     

Forward signaling mediated by ephrin-B3 prevents contralateral corticospinal axons from recrossing the spinal cord midline

To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling resulted in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract was defective as axons failed to respect the midline boundary of the spinal cord and bilaterally innervated both contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-B3 protein lacking its cytoplasmic domain did not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal corticospinal neurons as their fibers pathfind down the contralateral spinal cord. Our data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons.     

BRCA1-induced apoptosis involves inactivation of ERK1/2 activities

Mutation in the BRCA1 gene is associated with an increased risk of breast and ovarian cancer. Recent studies have shown that the BRCA1 gene product may be important in mediating responses to DNA damage and genomic instability. Previous studies have indicated that overexpression of BRCA1 can induce apoptosis or cell cycle arrest at the G(2)/M border in various cell types. Although the activation of JNK kinase has been implicated in BRCA1-induced apoptosis, the role of other members of the mitogen-activated protein kinase family in mediating the cellular response to BRCA1 has not yet been examined. In this study, we monitored the activities of three members of the MAPK family (ERK1/2, JNK, p38) in MCF-7 breast cancer cells and U2OS osteosarcoma cells after their exposure to a recombinant adenovirus expressing wild type BRCA1 (Ad.BRCA1). Overexpression of BRCA1 in MCF-7 cells resulted in arrest at the G(2)/M border; however, BRCA1 expression in U2OS cells induced apoptosis. Although BRCA1 induced JNK activation in both cell lines, there were marked differences in ERK1/2 activation in response to BRCA1 expression in these two cell lines. BRCA1-induced apoptosis in U2OS cells was associated with no activation of ERK1/2. In contrast, BRCA1 expression in MCF-7 cells resulted in the activation of both ERK1/2 and JNK. To directly assess the role of ERK1/2 in determining the cellular response to BRCA1, we used dominant negative mutants of MEK1 as well as MEK1/2 inhibitor PD98059. Our results indicate that inhibition of ERK1/2 activation resulted in increased apoptosis after BRCA1 expression in MCF-7 cells. Furthermore, BRCA1-induced apoptosis involved activation of JNK, induction of Fas-L/Fas interaction, and activation of caspases 8 and 9. The studies presented in this report indicate that the response to BRCA1 expression is determined by the regulation of both the JNK and ERK1/2 signaling pathways in cells.