Spanning Region Research Articles

Structural and functional characterization of AtPTR3, a stress-induced peptide transporter of Arabidopsis

A T-DNA tagged mutant line of Arabidopsis thaliana, produced with a promoter trap vector carrying a promoterless gus (uidA) as a reporter gene, showed GUS induction in response to mechanical wounding. Cloning of the chromosomal DNA flanking the T-DNA revealed that the insert had caused a knockout mutation in a PTR-type peptide transporter gene named At5g46050 in GenBank, here renamed AtPTR3. The gene and the deduced protein were characterized by molecular modelling and bioinformatics. Molecular modelling of the protein with fold recognition identified 12 transmembrane spanning regions and a large loop between the sixth and seventh helices. The structure of AtPTR3 resembled the other PTR-type transporters of plants and transporters in the major facilitator superfamily. Computer analysis of the AtPTR3 promoter suggested its expression in roots, leaves and seeds, complex hormonal regulation and induction by abiotic and biotic stresses. The computer-based hypotheses were tested experimentally by exposing the mutant plants to amino acids and several stress treatments. The AtPTR3 gene was induced by the amino acids histidine, leucine and phenylalanine in cotyledons and lower leaves, whereas a strong induction was obtained in the whole plant upon exposure to salt. Furthermore, the germination frequency of the mutant line was reduced on salt-containing media, suggesting that the AtPTR3 protein is involved in stress tolerance in seeds during germination.

Three-dimensional wake effects on flow-induced forces

Numerical simulations are carried out for a long slender rigid circular cylinder in a cross-flow to examine three-dimensional (3-D) wake effects on the flow-induced forces. The aim is to assess the validity and extent of the two-dimensional (2-D) assumption for both the mean drag and the flow-induced forces. In order to simulate the practical situation correctly, wall boundary conditions are specified at both ends of the cylinder. The long slender cylinder has different aspect ratios. A finite volume method (FVM) and a lattice Boltzmann method (LBM) are used to carry out the computations, and their results are compared with each other and with available experimental and simulation data. As a first attempt to assess the 2-D assumption, a Reynolds number Re = 100 and an aspect ratio a = 16 are chosen. At this Re and a, conventional experimental and numerical studies assume that the time-averaged flow is homogeneous and 2-D over a relatively large portion of the central span. However, present simulations indicate that vortex shedding from the nominally 2-D cylinder strongly depends on the span location and the flow-induced forces exhibit strong three dimensionality. The calculated mean drag and root-mean-square lift and drag vary greatly along the span. These results indicate that the 2-D assumption is not valid for the flow-induced forces, not even within a small region of the central span, for the aspect ratio examined. The validity and extent of the periodic boundary conditions assumption for a 3-D simulation of the flow and induced forces on a cylinder in a cross-flow is examined next. It is found that, within the range of a investigated, an appropriate period could not be found for the numerical simulation. The results further show that a has a significant effect on the calculated wake flow and the flow-induced forces.

Synthetic peptides derived from SARS coronavirus S protein with diagnostic and therapeutic potential

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important viral structural protein. Based on bioinformatics analysis, 10 antigenic peptides derived from the S protein sequence were selected and synthesized. The antigenicity and immunoreactivity of all the peptides were tested in vivo and in vitro. Four peptides (P6, P8, P9 and P10) which contain B cell epitopes of the S protein were identified, and P8 peptide was confirmed in vivo to have a potential in serological diagnosis. By using a syncytia formation model, we tested the neutralization ability of all 10 peptides and their corresponding antibodies. It is interesting to find that P8 and P9 peptides inhibited syncytia formation, suggesting that the P8 and P9 spanning regions may provide a good target for anti-SARS-CoV drug design. Our data suggest that we have identified peptides derived from the S protein of SARS-CoV, which are useful for SARS treatment and diagnosis.

Polycystin-1 Activates the Calcineurin/NFAT (Nuclear Factor of Activated T-cells) Signaling Pathway

Regulation of intracellular Ca(2+) mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway through Galpha(q) -mediated activation of phospholipase C (PLC). Transient transfection of HEK293T cells with an NFAT promoter-luciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3beta by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the Galpha(q) subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca(2+) channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFAT-green fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained elevation of intracellular Ca(2+) mediated by PC1 activation of Galpha(q) followed by PLC activation, release of Ca(2+) from intracellular stores, and activation of store-operated Ca(2+) entry, thus activating calcineurin and NFAT.

G Proteins, Plant Growth, and Parasite Invasion

The function of regulators of G protein signaling (RGSs) is just as the name indicates--in mammals and yeast, these proteins attenuate signals initiated by G protein-coupled receptors (GPCRs) at the cell surface. Chen et al. have now identified an RGS protein in Arabidopsis that functions together with a heterotrimeric G protein to control root and shoot growth. The plant RGS behaves biochemically like a bona fide family member, yet its sequence predicts a protein that contains a seven-transmembrane spanning region similar to GPCRs, quite unlike other known RGS proteins. The process of invasion of red blood cells by the malaria parasite Plasmodium falciparum is fundamental to disease progression. Harrison et al. reveal the requirement for GPCR activation in the host erythrocyte cell membrane to allow successful invasion. It is likely that the activated receptors promote the rearrangement of the rigid erythrocyte subcortical cytoskeleton to allow for the deformation of the membrane required during parasite invasion. J.-G. Chen, F. S. Willard, J. Huang, J. Liang, S. A. Chasse, A. M. Jones, D. P. Siderovski, A seven-transmembrane RGS protein that modulates plant cell proliferation. Science 301 , 1728-1731 (2003). [Abstract] [Full Text] T. Harrison, B. U. Samuel, T. Akompong, H. Hamm, N. Mohandas, J. W. Lomasney, K. Haldar, Erythrocyte G protein-coupled receptor signaling in malarial infection. Science 301 , 1734-1736 (2003). [Abstract] [Full Text]

Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica.

Adaptation of cells to acetic acid requires a hitherto unknown number of proteins. Studies on the GPR1 gene and its encoded protein in the ascomycetous fungus Yarrowia lipolytica have revealed an involvement of this protein in the molecular processes of adaptation to acetic acid. Gpr1p belongs to a novel family of conserved proteins in prokaryotic and eukaryotic organisms that is characterized by the two motifs (A/G)NPAPLGL and SYG(X)FW (GPR1_FUN34_YaaH protein family). Analysis of four trans-dominant mutations and N-terminal deletion analysis of Gpr1p identified the amino acid sequence FGGTLN important for function of this protein in Y. lipolytica. Deletion of GPR1 slowed down adaptation to acetic acid, but had no effect on growth in the presence of acetic acid. Expression of GPR1 is induced by acetic acid and moderately repressed by glucose. It was shown by subcellular fractionation that Gpr1p is an integral membrane protein, which is also suggested by the presence of five to six putative transmembrane spanning regions. Fluorescence microscopy confirmed a localization to the plasma membrane. A model is presented describing a hypothetical function of Gpr1p during adaptation to acetic acid.

How perennial are perennial plants?

Trade‐offs involving life span are important in the molding of plant life histories. However, the empirical examination of such patterns has so far been limited by the fact that information on life span is mainly available in terms of discrete categories; annuals, semelparous perennials and iteroparous perennials. We used transition matrix models to project continuous estimates of conditional life spans from published information on size‐ or stage‐structured demography for 71 perennial plant species. The projected life span ranged from 4.3 to 988.6 years and more than half of the species had a life span of more than 35 years. Woody plants had on average a projected life span more than four times as long as non‐woody plants. Life spans were higher in forests than in open habitats and individuals of non‐clonal species tended to have a longer life span than ramets of clonal species. Self‐incompatible plants on average lived longer than self‐compatible plants. There were no clear relations between life span and geographical region, dispersal syndrome, pollination mode, seed size or the presence of a seed bank. We conclude that accurate estimates of life span are central to understand how longevity is correlated to other traits within the group of perennial plants.

Characterization of a polymorphic Theileria annulata surface protein ( TaSP) closely related to PIM of Theileria parva: implications for use in diagnostic tests and subunit vaccines

Theileria annulata is a tick-transmitted protozoan that causes tropical theileriosis, an often fatal leukoproliferative disorder of cattle. To characterize and identify parasite proteins suitable as diagnostic antigens and/or vaccine candidates, a cDNA clone encoding a macroschizont stage protein was isolated and characterized (here designated TaSP). The gene, present as a single copy within the parasite genome, is transcribed in the sporozoite and schizont stage and codes for a protein of about 315 amino acids, having a predicted molecular weight of 36 kDa. Allelic variants were found within single parasite isolates and between isolates originating from different geographical regions. The N-terminal part contains a predicted signal peptide and the C-terminal section encodes membrane-spanning regions. Comparison of a number of cDNA clones showed that both these sequence regions are conserved while the central region shows both size and amino acid sequence polymorphism. High identity of the N- and C-terminal regions with the polymorphic immunodominant molecule (PIM) of Theileria parva (identity of 93%), the existence of a central polymorphic region and two short introns within genomic clones suggest that the presented gene/protein may be the T. annulata homologue of PIM. However, the central region of TaSP has no significant identity with PIM, contains no repetitive peptide motifs and is shorter, resulting in a lower molecular weight. The existence of the predicted secretion signal peptide and membrane spanning regions suggest that TaSP is located at the parasite membrane.

Demonstration of reduced in vivo surface expression of activating mutant thyrotrophin receptors in thyroid sections.

Thyroid function and growth are controlled by TSH. Hyperthyroidism can be due to Graves' Disease (GD), in which thyroid-stimulating antibodies mimic TSH, or gain-of-function mutations in the TSH receptor (TSHR). These activating mutations have poor surface expression when assessed in non-thyroidal cells in vitro but nothing is known of their in vivo behaviour. Several TSHR antibodies have been produced but none has been applied to thyroid paraffin sections. This study aimed to develop a technique suitable for use on paraffin sections and apply it to investigate TSHR expression in thyroids harbouring activating TSHR germline mutations compared with normal and GD thyroids. Immunocytochemistry coupled with antigen retrieval, using a spectrum of antibodies to the TSHR, was applied to paraffin sections of GD thyroid tissue. Subsequently, TSHR immunoreactivity was examined in three normal thyroids, three patients with GD and three patients with familial hyperthyroidism, due to different gain-of-function TSHR germline mutations, using the optimised protocol. Two antibodies, A10 and T3-495, to the extracellular domain (ECD) and membrane spanning region (MSR) of the TSHR respectively, produced specific basolateral staining of thyroid follicular cells. In normal and GD thyroids, basolateral staining with T3-495 was generally more intense than with A10, suggesting a possible surfeit of MSR over ECD. Graves' Disease thyroids have more abundant TSHR than normal glands. In contrast, thyroids harbouring gain-of-function mutations have the lowest expression in vivo, mirroring in vitro findings. The development of an immunocytochemical method applicable to paraffin sections has demonstrated that different molecular mechanisms causing hyperthyroidism result in the lowest (mutation) and highest (autoimmunity) levels of receptor at the thyrocyte surface.

Novel tropane-based irreversible ligands for the dopamine transporter.

3alpha-(diphenylmethoxy)tropane (benztropine) and its analogues are tropane ring-containing dopamine uptake inhibitors that display binding and behavioral profiles that are distinct from cocaine. We previously prepared a benztropine-based photoaffinity label [125I]-(N-[4-(4'-azido-3'-iodophenyl)butyl]-3alpha-[bis(4'-fluorophenyl)methoxy]tropane, [125I]1, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] 2 (RTI 82). To characterize further these different binding domains, photoaffinity ligands that had the 4'-azido-3'-iodophenyl substituent extended from the same position on the tropane ring were desirable. Thus, identification of the optimal alkyl linker between this substituent and the tropane nitrogen in the benztropine series was investigated to ultimately prepare the identical N-substituted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propyl] analogue of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthesized as well as two isothiocyanate analogues that do not require photoactivation (10a,b) for irreversible binding. The synthesis of these target compounds was achieved using a modification of the strategy developed for 1. Evaluation of these compounds for displacing [3H]WIN 35 428 binding at DAT in rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substituent, found in 1, provided optimal binding affinity and was chosen to replace the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothiocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesized. Both products bound to DAT with comparable potency (IC(50) = 30 nM) to RTI 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [3H]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These ligands will provide important tools for further characterizing the binding domains for tropane-based dopamine uptake inhibitors at the DAT.

Parametric study on multiple tuned mass dampers for buffeting control of Yangpu Bridge

A study on buffeting control of the Yangpu Bridge using a multiple tuned mass damper (MTMD) system is performed in this paper. The MTMD system consists of a set of TMDs which are attached to the center region of the bridge's main span and are symmetrical about the center of the main span as well as about the central line along the bridge span. It is found that the control efficiency of the MTMD system is sensitive to its frequency characteristics, namely, the central frequency ratio and the frequency bandwidth ratio. On the other hand, the damping ratio of the TMDs has less significant effects on the control efficiency. A total of seven MTMD systems with different mass ratios are designed. Each one of these seven MTMD systems can be used for the buffeting control of the Yangpu Bridge, depending on the required control efficiency and the available budget.

Genomic organization of the human GRIK2 gene and evidence for multiple splicing variants

Fast excitatory transmission in the vertebrate central nervous system is mediated mainly by l-glutamate. Here we present the genomic organization of the human GRIK2 gene, which codes for the kainate GluR6 receptor subunit, deduced from sequence data present in the public databases and analyzed by bioinformatic tools. By similarity search using the human GluR6 cDNA sequence against non-redundant databases, we found three positive entries (AP002528, AP002529, and AP002530 deposited by Hirakawa et al., 2000) which are part of a BAC contig of about 1 Mb spanning region 6q21. The GRIK2 gene was found to be split into 17 exons, covering about 670 kb of the region. The availability of the data on the genomic organization allowed the study of GRIK2 gene expression by RT-PCR analysis which was performed on human teratocarcinoma cell cultures (NT2) and on mRNA obtained from human hippocampus (Clontech). The study gives evidence for several different splicing variants in addition to the previously cloned human GluR6 cDNA (ID: U16126). The splicing mechanism leading to the different isoforms involves exons 11, 12 and 16. The mRNA containing exon 16 at the 3′ end is the homolog to the mouse GluR6-2. The translation of this mRNA would code for a different intracellular C-terminus, as compared to that coded by the known human isoform. The newly identify isoform is the predominant form expressed in human teratocarcinoma NT2 cells. All the newly identified mRNAs isoforms are expressed in NT2 cells and in human hippocampus mRNA at variable levels and would be responsible for the production of five different putative GluR6 receptor subunits, some differing in the C-terminal domains (mouse homolog) and some lacking specific transmembrane domains.

Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase.

The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyostelium discoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase.

Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase

The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyosteliumdiscoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase.

Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086-4094). We now report the cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. The heterologously expressed degradation pathway proteins are enzymatically active. Microcystinase (MlrA), the first enzyme in the degradative pathway, is a 336-residue endopeptidase, which displays only low sequence identity with a hypothetical protein from Methanobacterium thermoautotrophicum. Inhibition of microcystinase by EDTA and 1,10-phenanthroline suggests that it is a metalloenzyme. The most likely residues that could potentially chelate an active-site transition metal ion are in the sequence HXXHXE, which would be unique for a metalloproteinase. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442 residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2 family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402 residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the "penicillin-binding enzyme" family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.

Weibull modulus for diverse strength due to sample-specificity

By sample specificity it is meant that specimens with the same nominal material parameters and tested under the same environmental conditions may exhibit different behavior with diversified strength. Such an effect has been widely observed in the testing of material failure and is usually attributed to the heterogeneity of material at the mesoscopic level. The degree with which mesoscopic heterogeneity affects macroscopic failure is still not clear. Recently, the problem has been examined by making use of statistical ensemble evolution of dynamical system and the mesoscopic stress re-distribution model (SRD). Sample specificity was observed for non-global mean stress field models, such as the cluster mean field model, stress concentration at tip of microdamage, etc. Certain heterogeneity of microdamage could be sensitive to particular SRD leading to domino type of coalescence. Such an effect could start from the microdamage heterogeneity and then be magnified to other scale levels. This trans-scale sensitivity is the origin of sample specificity. The sample specificity leads to a failure probability Φ N with a transitional region 0< Φ N <1, so that globally stable and catastrophic modes could co-exist. It is found that the scatter in strength can fit the Weibull distribution very well. Hence, the Weibull modulus is indicative of sample specificity. Numerical results obtained from the SRD for different non-global mean stress fields show that Weibull modulus increases with increasing sample span and influence region of microdamage.

Effect of multiple serine/alanine mutations in the transmembrane spanning region V of the D2 dopamine receptor on ligand binding.

Three conserved serine residues (Ser193, Ser194, and Ser197) in transmembrane spanning region (TM) V of the D2 dopamine receptor have been mutated to alanine, individually and in combination, to explore their role in ligand binding and G protein coupling. The multiple Ser -->Ala mutations had no effect on the binding of most antagonists tested, including [3H]spiperone, suggesting that the multiple mutations did not affect the overall conformation of the receptor protein. Double or triple mutants containing an Ala197 mutation showed a decrease in affinity for domperidone, whereas Ala193 mutants showed an increased affinity for a substituted benzamide, remoxipride. However, dopamine showed large decreases in affinity (>20-fold) for each multiple mutant receptor containing the Ser193Ala mutation, and the high-affinity (coupled) state of the receptor (in the absence of GTP) could not be detected for any of the multiple mutants. A series of monohydroxylated phenylethylamines and aminotetralins was tested for their binding to the native and multiple mutant D2 dopamine receptors. The results obtained suggest that Ser193 interacts with the hydroxyl of S-5-hydroxy-2-dipropylaminotetralin (OH-DPAT) and Ser197 with the hydroxyl of R-5-OH-DPAT. We predict that Ser193 interacts with the hydroxyl of R-7-OH-DPAT and the 3-hydroxyl (m-hydroxyl) of dopamine. Therefore, the conserved serine residues in TMV of the D2 dopamine receptor are involved in hydrogen bonding interactions with selected antagonists and most agonists tested and also enable agonists to stabilise receptor-G protein coupling.

Mutations in the Interdomain Loop Region of thetetA(A) Tetracycline Resistance Gene Increase Efflux of Minocycline and Glycylcyclines

A novel class of tetracyclines, the glycylcyclines, have been shown to be active against bacterial strains harboring genes encoding tetracycline efflux pumps. However, two veterinary Salmonella isolates that carried tetracycline resistance determinants of the tetA(A) class were found to have reduced susceptibility to glycylcyclines, especially two early investigational glycylcyclines, DMG-MINO and DMG-DMDOT. These isolates were also quite resistant to tetracycline and minocycline. The isolates, one a strain of S. cholerasuis and the other, S. typhimurium, both carried the same novel tetA(A) variant, based on DNA sequencing, with one determinant plasmid encoded and the other located on the chromosome. This tetA(A) variant was cloned and shown to provide reduced susceptibility to the glycylcycline class although GAR-936, a glycylcycline currently in clinical development, was the least affected. The novel tetA(A) gene carries two mutations in the largest cytoplasmic loop of the efflux pump, which causes a double frameshift in codons 201, 202, and 203. This "interdomain region" of the efflux pump has generally been regarded as having no functional role in the efflux of tetracycline but the double frameshift is most likely responsible for the enhanced resistance observed and points to an interaction that was previously unrecognized. Mutants of the tetA(B) class with decreased susceptibility to the glycylcyclines were also generated in vitro. These all carried mutations in the portion of the tetA(B) gene encoding a transmembrane spanning region of the efflux pump. The laboratory-generated mutants point to the tight constraints in substrate recognition of the transmembrane-spanning region and may suggest that it will be the interdomain region of the pump that is likely to be the locus of future glycylcycline resistance mutations as these compounds enter clinical use.

Definition of a region of loss of heterozygosity at chromosome 11q in cervical carcinoma.

Chromosomal deletions at segment 11q23-q24 have been identified in a variety of human epithelial tumors, including cervical carcinoma (CC), indicating the presence in this region of at least a tumor suppressor gene (TSG) involved in the development of these neoplasms. To localize the 11q deletion target more precisely, 54 primary cervical carcinomas were examined for loss of heterozygosity (LOH) using a panel of microsatellite DNA markers mapping to 11p.15 and spanning region 11q23-qter. Nineteen tumors were found to have LOH at chromosome 11q. The highest frequency of LOH was observed at locus APOC-3, located in 11q23.1-q23.2, which was deleted in 42% of the informative cases. In contrast, LOH was infrequent at distal 11q in current series of CC. The smallest common region of loss included APOC-3 and was defined distally by marker D11S925 in region 11q23. The present data strongly suggest that the 11q suppressor gene(s) involved in cervical tumorigenesis is likely to be located at chromosome region 11q22-q23.

Identification and characterization of the human peroxin PEX3

The biogenesis of peroxisomes requires the interaction of several peroxins, encoded by PEX genes and is well conserved between yeast and humans. We have cloned the human cDNA of PEX3 based on its homology to different yeast PEX3 genes. The deduced peroxin HsPEX3 is a peroxisomal membrane protein with a calculated molecular mass of 42.1 kDa. We created N- and C-terminal tagged PEX3 to assay its topology at the peroxisomal membrane by immunofluorescence microscopy. Our results and the one predicted transmembrane spanning region are in line with the assumption that H sPEX3 is an integral peroxisomal membrane protein with the N-terminus inside the peroxisome and the C-terminus facing the cytoplasm. The farnesylated peroxisomal membrane protein PEX19 interacts with HsPEX3 in a mammalian two-hybrid assay in human fibroblasts. The physical interaction could be confirmed by coimmunoprecipitation of the two in vitro transcribed and translated proteins. To address the targeting of PEX3 to the peroxisomal membrane, the expression of different N- and C-terminal PEX3 truncations fused to green fluorescent protein (GFP) was investigated in human fibroblasts. The N-terminal 33 amino acids of PEX3 were necessary and sufficient to direct the reporter protein GFP to peroxisomes and seemed to be integrated into the peroxisomal membrane. The expression of a 1-16 PEX3-GFP fusion protein did not result in a peroxisomal localization, but interestingly, this and several other truncated PEX3 fusion proteins were also localized to tubular and/or vesicular structures representing mitochondria.