Amino-terminal Half Research Articles

Novel single chain antibodies to the prion protein identified by phage display

A well defined structure is available for the carboxyl half of the cellular prion protein (PrP c), while the structure of the amino terminal half of the molecule remains ill defined. The unstructured nature of the polypeptide has meant that relatively few of the many antibodies generated against PrP c recognise this region. To circumvent this problem, we have used a previously characterised and well expressed fragment derived from the amino terminus of PrP c as bait for panning a single chain antibody phage (scFv-P) library. Using this approach, we identified and characterised 1 predominant and 3 additional scFv-Ps that contained different V H and V L sequences and that bound specifically to the PrP c target. Epitope mapping revealed that all scFv-Ps recognised linear epitopes between PrP c residues 76 and 156. When compared with existing monoclonal antibodies (MAb), the binding of the scFvs was significantly different in that high level binding was evident on truncated forms of PrP c that reacted poorly or not at all with several pre-existing MAbs. These data suggest that the isolated scFv-Ps bind to novel epitopes within the amino-central region of PrP c. In addition, the binding of MAbs to known linear epitopes within PrP c depends strongly on the endpoints of the target PrP c fragment used.

NEDD1: Function in microtubule nucleation, spindle assembly and beyond

Nedd1 was originally identified as a developmentally regulated gene in the mouse central nervous system. NEDD1 has homologues across a range of species, being particularly conserved in a region of WD40 repeats contained in the amino-terminal half of the protein. Human NEDD1 was recently found to localise to the centrosome and mitotic spindle. It binds to the components of the γ-tubulin ring complex and target this complex to the centrosome and spindle. Depletion of NEDD1 causes loss of the γ-tubulin ring complex from the centrosome and results in the failure of microtubule nucleation and spindle assembly. In addition, phosphorylation of NEDD1 during mitosis is critical for targeting γ-tubulin to the spindle, but not the centrosome. There is still much unknown about the function of this protein and how it may be involved in development and disease. This short review summarises some of the recent work on NEDD1 and discusses how this interesting protein may have additional yet unexplored functions.

Green fluorescent protein as a reporter of prion protein folding

BackgroundThe amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP), as a method to examine the refolding of the amino terminal domain of mouse prion protein.ResultsFusion proteins of PrPc and GFP were expressed at high level in E.coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E.coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence.ConclusionUse of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction.

DNA immunization with 2C FMDV non-structural protein reveals the presence of an immunodominant CD8 +, CTL epitope for Balb/c mice

Outbreaks of foot and mouth disease virus (FMDV) have devastating economic consequences in affected areas. The presence of multiple serotypes and virus variants makes vaccination complicated. A better understanding of protective immune mechanisms may help in development of novel vaccines with cross protective capacity. While much attention has been devoted to humoral responses to FMDV, less is known about the role of cell-mediated responses in protective immunity. Predictions of potential CTL epitopes by two different computer algorithms identified the viral 2C protein as containing a potential murine H2-Kd CTL epitope located in its amino-terminal half. DNA vaccination of mice with a plasmid expressing the 2C protein and a fragment thereof confirmed that this was indeed a CTL epitope, as shown by interferon gamma (IFN-γ) induction in CD8 +, CD44 hi splenocytes after in vitro stimulation with peptides containing the amino acid sequence KYKDAKEWL, predicted for the CTL epitope. A peptide with the variant sequence KYKEAKEWL induced similar responses, indicating tolerability towards a conservative substitution at the altered residue. Virus infection likewise induced a measurable CTL response against KYKDAKEWL, although less clear due to a higher background of IFN-γ production in splenocytes from infected mice. Challenge of vaccinated mice showed that the CTL response induced by the 2C protein was not protective, since viremia and mortality were unaffected by vaccination. The implications for vaccine development are discussed in the context of cross-serotype reactive responses.

The Carboxyl-terminal Nucleoplasmic Region of MAN1 Exhibits a DNA Binding Winged Helix Domain

MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor beta superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.

Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.

Conductance of Connexin Hemichannels Segregates with the First Transmembrane Segment

Gap junction channels are intercellular channels that mediate the gated transfer of molecules between adjacent cells. To identify the domain determining channel conductance, the first transmembrane segment (M1) was reciprocally exchanged between Cx46 and Cx32E 143. The resulting chimeras exhibited conductances similar to that of the respective M1 donor. Furthermore, a chimera with the carboxy-terminal half of M1 in Cx46 replaced by that of Cx32 exhibited a conductance similar to that of Cx32E 143, whereas the chimera with only the amino-terminal half of M1 replaced retained the unitary conductance of wild-type Cx46. Extending the M1 domain swapping to other connexins by replacing the carboxy-terminal half of M1 in Cx46 with that of Cx37 yielded a chimera channel with increased unitary conductance close to that of Cx37. Furthermore, a point mutant of Cx46, with leucine substituted by glycine in position 35, displayed a conductance much larger than that of the wild type. Thus, the M1 segment, especially the second half, contains important determinants of conductance of the connexin channel.

The proline‐rich protein palladin is a binding partner for profilin

Palladin is an actin-associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin-binding proteins vasodilator-stimulated phosphoprotein (VASP), alpha-actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin-regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline-containing sequence in the amino-terminal half of palladin. Double-label immunofluorescence staining shows that palladin and profilin partially colocalize in actin-rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.

The Saccharomyces cerevisiae COQ10 Gene Encodes a START Domain Protein Required for Function of Coenzyme Q in Respiration

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q2. Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q6, the coq10 mutant has near normal amounts of Q6 in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic-rich protein family" Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q6/mol of protein. We propose that Coq10p is a Q6-binding protein and that in the coq10 mutant Q6 it is not able to act as an electron carrier, possibly because of improper localization.

The Carboxyl-Terminal Domain of Inducible Hsp70 Protects from Ischemic Injury in vivo and in vitro

Heat shock protein (Hsp)70 can suppress both necrosis and apoptosis induced by various injuries in vivo and in vitro. However, the relative importance of different functions and binding partners of Hsp70 in ischemic protection is unknown. To explore this question, we tested the ability of Hsp70-K71E, an adenosine triphosphate (ATP)ase-deficient point mutant, and Hsp70-381-640, a deletion mutant lacking the ATPase domain and encoding the carboxyl-terminal portion, to protect against ischemia-like injury in vivo and in vitro. Heat shock protein 70-wild type (-WT), -K71E, -381-640, and control vector plasmid LXSN were expressed in primary murine astrocyte cultures. Astrocytes overexpressing Hsp70-WT, -K71E, or -381-640 were all significantly protected from 4 h combined oxygen-glucose deprivation and 24 h reperfusion when assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or propidium iodide staining and cell counting (P < 0.05). Brains of rats were transfected with plasmids encoding Hsp70-WT, -K71E, -381-640, or LXSN 24 h before 2 h middle cerebral artery occlusion followed by 24 h reperfusion. Animals that overexpressed either of the mutant proteins or Hsp70-WT had significantly better neurological scores and smaller infarcts than control animals. Protection by both mutants was associated with reduced protein aggregation, as assessed by ubiquitin immunohistochemistry and reduced nuclear translocation of apoptosis-inducing factor. The results show that the carboxyl-terminal portion of Hsp70 is sufficient for neuroprotection. This indicates that neither the ability to fold denatured proteins nor interactions with cochaperones or other proteins that bind the amino-terminal half of Hsp70 are essential to ischemic protection.

Engineering of a Novel Saccharomyces cerevisiae Wine Strain with a Respiratory Phenotype at High External Glucose Concentrations

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Delta strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a non-ethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Delta strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain.

Detection of a Novelaph(2")Allele (aph[2"]-Ie) Conferring High-Level Gentamicin Resistance and a Spectinomycin Resistance Geneant(9)-Ia(aad9) in Clinical Isolates of Enterococci

Aminoglycoside-modifying enzymes (AMEs) are major factors that confer aminoglycoside resistance to enterococci. In an epidemiologic study on distribution of 12 AME genes in 534 recent clinical strains isolated from a Japanese hospital, two uncommon AME genes, ant(9)-Ia and a novel aph(2") allele, aph(2")-Ie, were detected. ant(9)-Ia had been reported only in Staphylococcus aureus and encodes spectinomycin adenylyltransferase ANT(9)-I, which confers resistance to spectinomycin. The ant(9)-Ia gene was detected in three strains, a single strain each of Enterococcus faecalis, E. faecium, and E. avium. Nucleotide sequences of ant(9)-Ia from these three enterococcal species were identical to that reported for S. aureus and considered to be located on Tn 554. The new aph(2") allele, designated aph(2")-Ie, was identified in three E. faecium strains. The aph(2")-Ie allele was genetically close to aph(2")-Id reported in E. casseliflavus (93.7% amino acid sequence identity; 96.3% similarity), while distant from aph(2")-Ia, aph(2")-Ib, or aph(2")-Ic (26.3-29.5% amino acid sequence identity). Sequence divergence between APH(2")-Id and APH(2")-Ie was mostly located in amino-terminal half. In contrast, sequences corresponding to the three motifs required for aminoglycoside phosphotransferase were conserved except for a single amino acid. Three E. faecium strains having aph(2")-Ie showed high-level resistance to gentamicin and streptomycin, but not to kanamycin, dibekacin, and tobramycin, unlike enzyme specificity described for aph(2")-Id in E. casseliflavus. Such a difference in resistance phenotype was suggested to be related to amino acid sequence divergence between APH(2")-Id and APH(2")-Ie.

Homo-oligomerization facilitates the interferon-antagonist activity of the ebolavirus VP35 protein

We have identified a putative coiled-coil motif within the amino-terminal half of the ebolavirus VP35 protein. Cross-linking studies demonstrated the ability of VP35 to form trimers, consistent with the presence of a functional coiled-coil motif. VP35 mutants lacking the coiled-coil motif or possessing a mutation designed to disrupt coiled-coil function were defective in oligomerization, as deduced by co-immunoprecipitation studies. VP35 inhibits signaling that activates interferon regulatory factor 3 (IRF-3) and inhibits (IFN)-α/β production. Experiments comparing the ability of VP35 mutants to block IFN responses demonstrated that the VP35 amino-terminus, which retains the putative coiled-coil motif, was unable to inhibit IFN responses, whereas the VP35 carboxy-terminus weakly inhibited the activation of IFN responses. IFN-antagonist function was restored when a heterologous trimerization motif was fused to the carboxy-terminal half of VP35, suggesting that an oligomerization function at the amino-terminus facilitates an “IFN-antagonist” function exerted by the carboxy-terminal half of VP35.

Roles of the C-terminal peptide binding domain of human inducible HSP70 in focal ischemic brain injury

The inducible heat shock protein 70 (Hsp70) is a molecular chaperone that is expressed primarily in response to stress, including heat shock, ischemia and glucose deprivation. Hsp70 consists of a 44 kDa amino terminal ATPase domain, an 18 kDa peptide or substrate binding domain, and a 10 kDa carboxy terminal domain. While a large number of proteins interact with Hsp70, only in some cases has the domain with which they interact been identified. Although it is accepted that Hsp70 can protect cells from ischemic injury, the mechanism is not known. Possible protective effects include prevention of protein aggregation, refolding denatured proteins and reduction of apoptosis. The relative importance of the ATP-binding domain compared to the peptide binding domain of HSP70 in ischemic protection is unknown. To explore this question we tested whether two Hsp70 mutant proteins could protect against ischemia-like injury of primary cultured murine astrocytes and reduce focal ischemic injury induced by transient (2 h) middle cerebral artery occlusion (MCAO). The two mutants studied were 1) Hsp70 K71E, a point mutation that abrogates ATP binding and renders the protein deficient in folding ability and 2) Hsp70 381-640 a deletion mutant lacking the ATP binding domain. Hsp70 wild type (WT), -K71E, -381-640 and vector plasmid LXSN were expressed in primary murine astrocyte cultures using retroviral mediated transfection. After G418 selection, more than 95% of cells expressed these proteins by immunostaining. Cultures were then subjected to combined oxygen-glucose deprivation (OGD) and cell survival was assessed by MTT assay or PI staining and cell counting. Astrocytes overexpressing Hsp70 -WT, -K71E or -381-640 were all significantly protected from 4 h OGD (cell viability was increased about 50%, p < 0.05). We then tested the ability of these mutant proteins to protect rats from transient focal ischemic injury. Plasmids encoding Hsp70 -K71E, -381-640, or the control backbone plasmid LXSN were stereotactically injected into the left lateral ventricle 24 h prior to MCAO. These mutant proteins were expressed in both astrocytes and neurons as determined by double immunostaining. Animals that overexpressed either of these two mutant proteins had significantly better neurological scores and smaller infarct volumes (30-35%) assessed at 24 hr (infarction volume (mm3): 233.538.6 in control group; 152.2543.95 in Hsp70 K71E group; and 162.7132.5 in Hsp70 381-640 group, P<0.05 compared to control, n=8 in each group). Protection by both mutants was associated with reduced protein aggregation and apoptosis, as assessed by ubiquitin immunohistochemistry and Klenow staining. Both the point mutant and the deletion mutant were found to reduce ischemic injury. Since the carboxy terminal half of the protein is sufficient for protection, interaction with protein partners that bind the aminoterminal half of the protein are not essential to ischemic protection. Also, since neither mutant can facilitate folding, their efficacy against ischemia suggests that inhibiting protein aggregation is sufficient to reduce ischemic injury and apoptotic cell death.

Saposin C: Neuronal Effect and CNS Delivery by Liposomes

Saposin C is one of four small lipid-binding proteins that derive from a single precursor protein, named prosaposin (PSAP). PSAP has several neuronal effects, including neurite outgrowth stimulation, neuron preservation, and nerve regeneration enhancement. A minimal domain required for PSAP's neurotrophic function is located in the amino-terminal half of saposin C. Genetic defects of the PSAP gene in humans and mice lead to a complex lysosomal storage disease. The skin fibroblasts from PSAP- and saposin C-deficient patients have a massive accumulation of multivesicular bodies (MVBs). Incorporation of exogenous saposin C-containing liposomes into the cultured PSAP-/- cells reduced the accumulated MVBs to normal levels. Internalized saposin C was localized to late endosomes and lysosomes. MVBs are crucial for maintaining the cellular homeostasis required for neuronal development and growth. PSAP-/- mice have a short life span (30 days) and central nervous system (CNS) neuronal degeneration. Similar to PSAP-/- fibroblasts, excessive MVBs accumulated in CNS neurons and brain tissues of PSAP-null mice. Cultured cortical and hippocampal neurons from PSAP-/- mice had poor survival and displayed a neurite degenerative pattern. Delivery of saposin C ex vivo into cultured neurons and in vivo into brain neuronal cells in mice across the blood-brain barrier was accomplished with intravenously administered dioleoylphosphatidylserine (DOPS) liposomes. These studies may yield a new therapeutic approach for neuron protection, preservation, and regeneration.

Psc3 cohesin of Schizosaccharomyces pombe: cell cycle analysis and identification of three distinct isoforms

Cohesins are a group of proteins that function to mediate correct chromosome segregation, DNA repair and meiotic recombination. This report presents the amino acid sequence for the Schizosaccharomyces pombe cohesin Psc3 based on the translation of the cDNA sequence, showing that the protein is smaller than previously predicted. Interestingly, comparison of the amino acid and DNA coding sequences of Psc3 with fission yeast Rec11 meiotic region-specific recombination activator shows that both intron positioning within the genes and the amino-terminal half of the two proteins are highly conserved. We demonstrate that although the intergenic region upstream of the psc3+ start codon contains a consensus sequence for the cell-cycle regulatory MluI cell-cycle box, psc3+ transcription is not differentially regulated during the mitotic cell cycle. Finally, we demonstrate that an epitope-tagged version of Psc3 undergoes no major changes during the mitotic cell cycle. However, instead we identify at least three distinct isoforms of Psc3, suggesting that post-translational modification of Psc3 contributes to the regulation of cohesion function.

CLONING AND NUCLEOTIDE SEQUENCE OF A FULL-LENGTH cDNA ENCODING ASCARIS SUUM PHOSPHOFRUCTOKINASE

The nucleotide sequence of a full-length cDNA encoding phosphofructokinase (PFK) enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2,653 bases comprises a single open reading frame of 2,452 bases and a noncoding region of 201 bases after the stop codon. The mature protein contains 812 amino acids and has a molecular mass of 90,900 Da. The amino acid sequences of several peptides derived from the purified protein show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the protein with those of 3 other worms as well as those of human, rabbit, and bacterial enzymes reveals highly conserved regions interrupted with stretches of lesser sequence similarity. Analyses of the subunit primary structure reveal, as in other eukaryotic PFKs, that the amino-terminal half is homologous to the carboxy-terminal half, supporting the hypothesis that the PFK gene evolved by duplication of the prokaryotic gene and that the allosteric sites arose by mutations at the catalytic site. The location of the phosphorylation site is unique and different compared with other PFKs and plays a key role in regulation of the enzyme activity. Structural motifs such as the putative substrate and effector binding domains and also the key amino acids involved therein are clearly identified by alignment of all the PFK protein sequences.

Mutational Analysis of the Stator Subunit E of the Yeast V-ATPase

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.

USING TUMOR MARKERS TO PREDICT THE SURVIVAL OF PATIENTS WITH METASTATIC RENAL CELL CARCINOMA

Approximately 30% of renal cell carcinomas (RCCs) present as metastatic disease. Molecular markers have the potential to characterize accurately the biological behavior of tumors and they may be useful for determining prognosis. A custom tissue array was constructed using clear cell RCC from 150 patients with metastatic RCC who underwent nephrectomy prior to immunotherapy. The tissue array was stained for 8 molecular markers, namely Ki67, p53, gelsolin, carbonic anhydrase (CA)9, CA12, PTEN (phosphatase and tensin homologue deleted on chromosome 10), epithelial cell adhesion molecule and vimentin. Marker status and established clinical predictors of prognosis were considered when developing a prognostic model for disease specific survival. On univariate Cox regression analysis certain markers were statistically significant predictors of survival, namely CA9 (p <0.00001), p53 (p = 0.0072), gelsolin (p = 0.030), Ki67 (p = 0.036) and CA12 (p = 0.043). On multivariate Cox regression analysis that included all markers and clinical variables CA9 (p = 0.00002), PTEN (p <0.0001), vimentin (p = 0.0032), p53 (p = 0.028), T category (p = 0.0025) and performance status (p = 0.0013) were significant independent predictors of disease specific survival and they were used to construct a combined molecular and clinical prognostic model. The bias corrected concordance index (C-index) of this combined prognostic model was C = 0.68, which was significantly higher (p = 0.0033) than that of a multivariate clinical predictor model (C = 0.62) based on the UCLA Integrated Staging System (T category, histological grade and performance status). In patients with clear cell RCC a prognostic model for survival that includes molecular and clinical predictors is significantly more accurate than a standard clinical model using the combination of stage, histological grade and performance status.

The Plowright vaccine strain of Rinderpest virus has attenuating mutations in most genes

The currently used vaccine strain of Rinderpest virus was derived by serial passage of the highly virulent Kabete 'O' strain (KO). A full-length cDNA copy of the KO strain was made from which a virus identical in pathogenicity to the wild-type virus was rescued. A series of chimeric viruses was prepared in which the coding sequences for the N, P, F, H or L proteins were replaced with the corresponding sequences from the vaccine strain. The KO-based virus with the vaccine strain H gene and that with the carboxy-terminal half of the L gene replaced with the corresponding sequence from the vaccine strain retained all or almost all of the virulence of the original KO virus. Animals infected with the KO-based virus containing the vaccine strain N, P or F gene, or the amino-terminal half of the L gene, developed high and prolonged pyrexia and leukopenia, but with reduced or absent lesions and other clinical signs; although partially attenuated, none was nearly as attenuated as the vaccine strain itself. These data indicate that the high attenuation and stability of the current vaccine are due to the accumulation of a number of separate mutations, none of which is itself so sufficiently debilitating that there is strong selective pressure in favour of the revertant.