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Biomedical subjects

E W Jones

Publications and source records attributed to E W Jones.

At least 37 records · Page 2Linked to original sources
PubMed1994

The yeast homolog of H < beta > 58, a mouse gene essential for embryogenesis, performs a role in the delivery of proteins to the vacuole.

The highly conserved and ubiquitously expressed mouse gene H < beta > 58, identified through insertional mutagenesis, has been shown to be essential for early postimplantation development in mouse, but the mechanism by which it acts is unknown (Radice et al. 1991; Lee et al. 1992). We report here the identification of a yeast gene related to the H < beta > 58 gene and provide biochemical and genetic evidence for its function within the cell. The gene, PEP8, plays a role in the delivery of proteins to the vacuole. Disruption of the gene did not affect cell viability. However, the disruptants were shown to have a defect in the processing of the soluble vacuolar proteases but not the membrane vacuolar hydrolases. The processing defect appeared to be a consequence of the inability of the soluble vacuolar hydrolase to reach the vacuole. Although a small amount of the vacuolar precursors was mis-sorted to the extracellular medium, mis-sorting did not appear to be the primary defect in these cells. Pep8p was identified by epitope tagging of the protein. Biochemical fractionation indicated that the protein was peripherally bound to membranes. Immuno-gold electron microscopy indicated that the Pep8p localized to vacuolar membranes. Complementation experiments with the mouse H < beta > 58 cDNA revealed that a Pep8p-H < beta > 58 fusion protein in which the carboxy-terminal 85 amino acids of Pep8p were replaced by the carboxy-terminal 115 amino acids of H < beta > 58 was functional.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1994

STV1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p.

The Saccharomyces cerevisiae gene, VPH1 (Vacuolar pH 1), encodes a 95-kDa integral membrane subunit of the vacuolar-type H(+)-ATPase (V-ATPase) that is required for enzyme assembly; disruption of the VPH1 gene impairs vacuolar acidification (Manolson, M.F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J. Biol. Chem. 267, 14294-14303). Here we show that STV1 (Similar To VPH1) encodes an integral membrane polypeptide of 102 kDa with 54% identity with the peptide sequence of Vph1p. High copy expression of STV1 partially restores vacuolar acidification in a delta vph1 mutant strain; solubilization and fractionation of membrane proteins from these vacuoles show that Stv1p co-purifies with bafilomycin A1-sensitive ATPase activity and with the 60- and 69-kDa V-ATPase subunits. Immunofluorescence microscopy of strains bearing a single copy of epitope-tagged STV1 reveals punctate staining of the cytoplasm; overexpression of epitope-tagged Stv1p reveals both punctate cytoplasmic staining and vacuolar membrane staining. Northern analysis shows that disruption of STV1 does not affect the level of transcription of VPH1 and that disruption of VPH1 does not affect the level of transcription of STV1. Strains bearing disruption of genes encoding other V-ATPase subunits (VMA1, VMA2, VMA3, and VMA4) fail to grow on media supplemented with 100 mM CaCl2 or 4 mM ZnCl2, media buffered to pH 7.5, or media with a glycerol carbon source. On the same types of media only a delta vph1 delta stv1 double disruption mutant has growth phenotypes equivalent to strains bearing a single disruption of the VMA1, VMA2, VMA3, and VMA4 genes; a delta vph1 strain has only moderate growth inhibition while a delta stv1 strain has wild type growth on the conditions listed above. We conclude that Stv1p is a functional homologue of Vph1p and suggest that Stv1p and Vph1p may be equivalent subunits for V-ATPases located on different organelles. The function of these 100-kDa homologues may be to target or regulate other common V-ATPase subunits for two distinct cellular locations.

Amino Acid Sequence↗
PubMed1994

Desmoplastic malignant melanoma. An immunocytochemical study of 25 cases.

Using a panel of seven cell markers, we studied the value of immunocytochemical labelling in the histological diagnosis of desmoplastic malignant melanoma. Sections from routine formalin-fixed tissue of 45 surgical specimens were obtained from 25 cases of malignant melanoma that showed well-marked desmoplastic or neurotropic features. Routinely stained sections (Haematoxylin- and -eosin and melanin stains) were compared with the following panel of seven antibodies: S-100, neuron-specific enolase (NSE), vimentin, factor XIIIa (FXIIIa), desmin and the newer, supposedly more specific antimelanoma antibodies HMB45 and NKIC3. S-100 and NSE were the most sensitive antibodies for desmoplastic malignant melanoma with strong labelling of spindle cells in most cases. In contrast, results for NKIC3 were more variable; results were negative in nearly half the tumours, but strong labelling was seen in six cases (27%). Positive labelling for HMB45 was noted in five tumours (22%); it was mostly confined to small groups of cells in the superficial part of the lesions. Tumour spindle cells were negative for FXIIIa in all cases; there was no increase in the number of positive dermal dendritic cells compared to conventional and spindle cell melanoma. All tumours were desmin-negative, but most were vimentin-positive. Our findings indicate that immunocytochemistry is of less value in the diagnosis of desmoplastic malignant melanoma that it is with other types of malignant melanoma. However, positive or negative labelling for S-100 protein and NSE is useful for suggesting or excluding a diagnosis of desmoplastic malignant melanoma; neither marker is specific and, in particular, positive labelling is also found in most neurofibromas and benign cellular naevi.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Mitochondrial mutation commonly associated with Leber's hereditary optic neuropathy observed in a patient with Wolfram syndrome (DIDMOAD).

DIDMOAD is usually considered an autosomal recessive condition, with wide phenotypic variation, but the possibility of mitochondrial mutations occurring in this condition has been considered. A 19 year old man presented with long standing diabetes mellitus, optic atrophy, and grand mal seizures. Further investigations showed unilateral sensorineural hearing loss and the most common mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy, which was inherited from his mother. This suggests the DIDMOAD phenotype is a mitochondrial disorder in some cases and is likely to have a heterogeneous aetiology.

Adult↗
PubMed1993

Phenotypic analysis of proteinase A mutants. Implications for autoactivation and the maturation pathway of the vacuolar hydrolases of Saccharomyces cerevisiae.

We have isolated a number of mutants deficient in activity of the vacuolar hydrolase proteinase A (PrA). The mutations were sequenced and although they all map in the PEP4 gene, which encodes the precursor to PrA, three distinguishable phenotypes have surfaced. The properties of the pep4-7 missense mutant suggested that the activation of the precursor to proteinase A is due to an autocatalytic cleavage. PrA active site mutations were constructed and resulted in accumulation of PrA antigen in the inactive precursor form. Although protease B (PrB), another vacuolar hydrolase, is not required for the production of active PrA, the active form of PrA that accumulates in a strain lacking PrB is larger than that found in a strain containing active PrB. We have purified this larger form of PrA and determined that it bears 7 additional amino acids at its NH2 terminus. It has become apparent from all the studies performed on the maturation pathway of the vacuolar hydrolases that there is a great deal of redundancy built into the system.

Amino Acid Sequence↗
PubMed1993

The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H(+)-ATPase.

Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc-sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1.2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open reading frame that encoded a protein of 215 amino acids. Computer analysis indicated that the protein, which has a predicted molecular mass of 25,286 Daltons, has two distinct membrane-spanning domains. Biochemical studies indicated that strains bearing the vph2 mutation have greatly reduced levels of vacuolar proton pumping and ATPase activity and that the nucleotide binding subunits of the multimeric vacuolar H(+)-ATPase failed to be correctly targeted to the vacuolar membrane. The vph2 mutant fails to grow on YEP glycerol medium and on media containing 100 mM-CaCl2 or 4 mM-ZnCl2 or buffered to pH 7.5, a phenotype observed in strains carrying deletions in the genes encoding several vacuolar H(+)-ATPase subunits. The VPH2 gene is identical to the VMA12 gene (T. Stevens and Y. Anraku, personal communication).

Alkaline Phosphatase↗
PubMed1993

Desmoplastic malignant melanoma: a clinicopathological study of 25 cases.

Sixteen cases of malignant melanoma which showed prominent desmoplastic and/or neurotropic features occurring throughout the tumour were compiled from the St John's Dermatology Centre histopathological archives. A further nine melanomas in which both conventional and desmoplastic melanoma were present concomitantly were also studied (three tumours with 66% desmoplastic change, two with 50%, and four with less than 50%). There were 14 males and 11 females, with a mean age of 64 years (range 39-86). The mean interval between presentation and diagnosis was 8 months. Eighteen of the 25 tumours were located on the head and neck, three were on the trunk, one was on the upper limb and three were on the lower limb. Histological review revealed 21 of 25 tumours with overlying atypical lentiginous hyperplasia, lentigo maligna melanoma, or superficial spreading malignant melanoma. Neurotropism was present in nine tumours, with the changes confined to local recurrences in two instances; neuroid differentiation was present in four tumours, and neural and perineural tumour spread was present in four tumours. The depth of invasion exceeded 6 mm in seven tumours, and was 2-6 mm in 16, and less than 2 mm in two. Eighteen of the 25 tumours were incompletely excised at the time of the first excision. Lymphoid aggregates were present in 16 tumours, but in most cases were limited to a few lymphoid foci. Melanin was identified in the dermal component of only five tumours, but not in areas showing typical histological features of desmoplastic malignant melanoma. Treatment was by surgical excision in all cases, and was preceded by radiotherapy in one case. Details of follow-up were obtained in all cases, and the duration ranged from 9 months to 10 years (mean, 3 years 11 months). Eleven patients had died; nine from melanoma and two from other causes. One patient was alive, with deep, inoperable local recurrence. Thirteen patients were alive and clinically free from tumour, including two patients in whom there had been local recurrence. A lower rate of neurotropism was present in the nine patients with partial desmoplastic change compared with those with desmoplasic change throughout the tumour, and represented the only significant difference between the two groups of patients.

Adult↗
PubMed1992

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

Amino Acid Sequence↗
PubMed1992

Genes required for vacuolar acidity in Saccharomyces cerevisiae.

Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein. Thirty nine vacuolar pH (Vph-) mutants were identified. Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes. The remaining mutants defined eight complementation groups of vph mutations. No alleles of the VAT2 or TFP1 genes (known to encode subunits of the vacuolar H(+)-ATPase) were identified in the Vph- screen. Strains bearing mutations in any of six of the VPH genes failed to grow on medium buffered at neutral pH; otherwise, none of the vph mutations caused notable growth inhibition on standard yeast media. Expression of the vacuolar protease, carboxypeptidase Y, was defective in strains bearing vph4 mutations but was apparently normal in strains bearing any of the other vph mutations. Defects in vacuolar morphology at the light microscope level were evident in all Vph- mutants. Strains that contained representative mutant alleles of the 17 previously described PEP genes were assayed for vacuolar pH; mutations in seven of the PEP genes (including PEP3, PEP5, PEP6 and PEP7) caused loss of vacuolar acidity.

Alleles↗
PubMed1992

Intravascular spread of keratoacanthoma. An alarming but benign phenomenon.

We report a patient with a keratoacanthoma of the scalp in which there was invasion of several medium-sized vessels by the tumour at a distance from the main lesion. A marked inflammatory response within the invaded vessels as well as a benign clinical course do not suggest that this phenomenon represents malignant transformation.

Blood Vessels↗
PubMed1992

The quality and relevance of peripheral neuropathy data on a diabetic clinical information system.

Routinely collected peripheral neuropathy data entered on a diabetic clinical information system since 1979 have been audited for completeness, consistency, accuracy (inter-observer variation), validity by comparison with biothesiometry, and relevance by life table analysis for foot ulceration. Peripheral neuropathy was defined by a neuropathy disability score > or = 4. The data were 98% complete. Forty-nine of 3405 (1.4%) had inconsistent records. Agreement between observers for clinical examination was significant (p < 0.05) for aggregate neuropathy score and its individual components except the knee jerk: Kappa score for observer variation for neuropathy score 0.56 (95% confidence interval 0.36-0.76). There was good agreement between neuropathy defined as aggregate score > or = 4, and as combined vibration perception thresholds for both feet > 60 V: Kappa statistic 0.62 (95% confidence interval 0.44-0.80). The chance of developing a foot problem in 3 years increased from 3% for patients with a score of zero to 45% for people with a score of between 9 and 12. We conclude that the calculation of a clinical neuropathy score is a simple, valid and relevant method for diabetes care both in hospital and the community. When combined with palpation of peripheral pulses most patients at risk of foot ulceration can be identified allowing targeting of preventive chiropody and orthotic resources.

Diabetic Neuropathies↗
PubMed1992

Evidence for a conserved 95-120 kDa subunit associated with and essential for activity of V-ATPases.

Vacuoles purified from Saccharomyces cerevisiae bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. Furthermore, the vacuolar H(+)-ATPase (V-ATPase) nucleotide binding subunits were no longer associated with vacuolar membranes yet were present at wild-type levels in yeast whole-cell extracts. The VPH1 gene was cloned by screening a lambda gt11 expression library with antibodies directed against a 95 kDa vacuolar integral membrane protein and independently cloned by complementation of the vph1-1 mutation. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is required for vacuolar H(+)-ATPase assembly and vacuolar acidification but is not essential for cell viability or for targeting and maturation of vacuolar proteases. VPH1 encodes a predicted polypeptide of 840 amino acid residues (95.6 kDa) with putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with V-ATPase activity. Vph1p has 42% identity to the 116 kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, 42% identity to the TJ6 mouse immune suppressor factor, 42% identity to the Caenorhabditis elegans proton pump homologue and 54% identity to the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene.

Adenosine Triphosphatases↗
PubMed1991

Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence.

Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae. It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C. M., Dixon, C. K., and Jones, E. W. (1989) J. Cell Biol. 108, 309-324). This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed. To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis. The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism. The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic. A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB. Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A. The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.

Base Sequence↗