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Biomedical subjects

D W Andrews

Publications and source records attributed to D W Andrews.

At least 19 recordsLinked to original sources
PubMed2005

Fractionated stereotactic radiotherapy for parasellar meningiomas: a preliminary report of visual outcomes.

BACKGROUND/AIM: Fractionated stereotactic radiotherapy (FSRT) is a new treatment for brain tumours that are close to critical structures, such as the visual apparatus. This study aims to assess the visual outcomes for patients with parasellar meningioma following FSRT. METHODS: A retrospective, non-comparative case series of 13 patients with parasellar meningiomas who were treated in one institution with FSRT between January 1995 and January 2001. RESULTS: 13 patients (26 eyes) were followed for a mean of 2 years. Visual acuity improved in four eyes (12.5%), remained stable in 18 eyes (75%), and worsened in three eyes (12.5%). Visual field improved in 15 eyes (57%), remained stable in six eyes (23%), and worsened in four eyes (15%). No adverse visual outcome occurred as a result of radiation. CONCLUSION: These preliminary findings suggest that FSRT is a safe and effective treatment for parasellar meningiomas.

Adult↗
PubMed2001

Cytoplasmic O-glycosylation prevents cell surface transport of E-cadherin during apoptosis.

Cellular adhesion is regulated by members of the cadherin family of adhesion receptors and their cytoplasmic adaptor proteins, the catenins. Adhesion complexes are regulated by recycling from the plasma membrane and proteolysis during apoptosis. We report that in MCF-7, MDA-MB-468 and MDCK cells, induction of apoptosis by agents that cause endoplasmic reticulum (ER) stress results in O-glycosylation of both beta-catenin and the E-cadherin cytoplasmic domain. O-glycosylation of newly synthesized E-cadherin blocks cell surface transport, resulting in reduced intercellular adhesion. O-glycosylated E-cadherin still binds to beta- and gamma-catenin, but not to p120-catenin. Although O-glycosylation can be inhibited with caspase inhibitors, cleavage of caspases associated with the ER or Golgi complex does not correlate with E-cadherin O-glycosylation. However, agents that induce apoptosis via mitochondria do not lead to E-cadherin O-glycosylation, and decrease adhesion more slowly. In MCF-7 cells, this is due to degradation of E-cadherin concomitant with cleavage of caspase-7 and its substrate poly(ADP-ribose) polymerase. We conclude that cytoplasmic O-glycosylation is a novel, rapid mechanism for regulating cell surface transport exploited to down-regulate adhesion in some but not all apoptosis pathways.

Animals↗
PubMed2001

Stereotactic radiosurgery and fractionated stereotactic radiotherapy for the treatment of acoustic schwannomas: comparative observations of 125 patients treated at one institution.

BACKGROUND: Stereotactic radiosurgery (SRS) and, more recently, fractionated stereotactic radiotherapy (SRT) have been recognized as noninvasive alternatives to surgery for the treatment of acoustic schwannomas. We review our experience of acoustic tumor treatments at one institution using a gamma knife for SRS and the first commercial world installation of a dedicated linac for SRT. METHODS: Patients were treated with SRS on the gamma knife or SRT on the linac from October 1994 through August 2000. Gamma knife technique involved a fixed-frame multiple shot/high conformality single treatment, whereas linac technique involved daily conventional fraction treatments involving a relocatable frame, fewer isocenters, and high conformality established by noncoplanar arc beam shaping and differential beam weighting. RESULTS: Sixty-nine patients were treated on the gamma knife, and 56 patients were treated on the linac, with 1 NF-2 patient common to both units. Three patients were lost to follow-up, and in the remaining 122 patients, mean follow-up was 119 +/- 67 weeks for SRS patients and 115 +/- 96 weeks for SRT patients. Tumor control rates were high (> or =97%) for sporadic tumors in both groups but lower for NF-2 tumors in the SRT group. Cranial nerve morbidities were comparably low in both groups, with the exception of functional hearing preservation, which was 2.5-fold higher in patients who received conventional fraction SRT. CONCLUSION: SRS and SRT represent comparable noninvasive treatments for acoustic schwannomas in both sporadic and NF-2 patient groups. At 1-year follow-up, a significantly higher rate of serviceable hearing preservation was achieved in SRT sporadic tumor patients and may therefore be preferable to alternatives including surgery, SRS, or possibly observation in patients with serviceable hearing.

Adult↗
PubMed2001

FtsY binds to the Escherichia coli inner membrane via interactions with phosphatidylethanolamine and membrane proteins.

Targeting of many polytopic proteins to the inner membrane of prokaryotes occurs via an essential signal recognition particle-like pathway. FtsY, the Escherichia coli homolog of the eukaryotic signal recognition particle receptor alpha-subunit, binds to membranes via its amino-terminal AN domain. We demonstrate that FtsY assembles on membranes via interactions with phosphatidylethanolamine and with a trypsin-sensitive component. Both interactions are mediated by the AN domain of FtsY. In the absence of phosphatidylethanolamine, the trypsin-sensitive component is sufficient for binding and function of FtsY in the targeting of membrane proteins. We propose a two-step mechanism for the assembly of FtsY on the membrane similar to that of SecA on the E. coli inner membrane.

Bacterial Proteins↗
PubMed2001

Results of a pilot study involving the use of an antisense oligodeoxynucleotide directed against the insulin-like growth factor type I receptor in malignant astrocytomas.

PURPOSE: Preclinical animal experiments support the use of an antisense oligodeoxynucleotide directed against the insulin-like growth factor type I receptor (IGF-IR/AS ODN) as an effective potential antitumor agent. We performed a human pilot safety and feasibility study using an IGF-IR/AS ODN strategy in patients with malignant astrocytoma. PATIENTS AND METHODS: Autologous glioma cells collected at surgery were treated ex vivo with an IGF-IR/AS ODN, encapsulated in diffusion chambers, reimplanted in the rectus sheath within 24 hours of craniotomy, and retrieved after a 24-hour in situ incubation. Serial posttreatment assessments included clinical examination, laboratory studies, and magnetic resonance imaging scans. RESULTS: Other than deep venous thrombosis noted in some patients, no other treatment-related side effects were observed. IGF-IR/AS ODN-treated cells, when retrieved and assessed, were < or = 2% intact by trypan blue exclusion, and none of the intact cells were viable in culture thereafter. Parallel Western blots disclosed IGF-IR downregulation to < or = 10% after ex vivo antisense treatment. At follow-up, clinical and radiographic improvements were observed in eight of 12 patients, including three cases of distal recurrence with unexpected spontaneous or postsurgical regression at either the primary or the distant intracranial site. CONCLUSION: Ex vivo IGF-IR/AS ODN treatment of autologous glioma cells induces apoptosis and a host response in vivo without unusual side effects. Subsequent transient and sustained radiographic and clinical improvements warrant further clinical investigations.

Adult↗
PubMed2001

Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event.

The disruption of mitochondrial function is a key component of apoptosis in most cell types. Localization of Bcl-2 to the outer mitochondrial and endoplasmic reticulum membranes is consistent with a role in the inhibition of many forms of apoptosis. In Rat-1 cells, a Bcl-2 mutant targeted exclusively to the endoplasmic reticulum (Bcl-cb5) was effective at inhibiting apoptosis induced by serum starvation/myc, or ceramide but not apoptosis induced by etoposide. The former conditions cause a decrease in mitochondrial transmembrane potential (Deltapsi(m)) as an early event that precedes the release of cytochrome c from mitochondria. By contrast, when cells are exposed to etoposide, a situation in which cytochrome c release and membrane localization of the pro-apoptotic protein Bax precede loss of Deltapsi(m), wild type Bcl-2 but not Bcl-cb5 prevents apoptosis. Therefore, Bcl-2 functions in spatially distinct pathways of apoptosis distinguished by the order of cytochrome c release and loss of Deltapsi(m).

Amino Acid Chloromethyl Ketones↗
PubMed2001

Cytochrome-C localizes in secretory granules in pancreas and anterior pituitary.

We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome-c in normal rat tissues, employing a wide variety of monoclonal and polyclonal antibodies against mammalian cytochrome-c. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows highly specific labeling of mitochondria in all tissues examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen and thyroid. In pancreatic acinar cells and anterior pituitary, however, there was also strong cytochrome-c reactivity in zymogen granules and growth hormone granules, respectively. In the pancreas, strong immunoreactivity is also detected in condensing vacuoles and in the acinar lumen. Immunocytochemical controls included (i) use of monoclonal antibodies to horse cytochrome-c which recognize an epitope not present in rat cytochrome-c, (ii) preadsorption of antibodies with purified cytochrome-c, and (iii) omission of the primary antibody. The indicated presence of cytochrome-c outside mitochondria in certain tissues under normal physiological conditions raises interesting questions concerning translocation mechanisms and the cellular functions of cytochrome-c.

Animals↗
PubMed2001

Proficiency in neurosurgery.

This evaluation of proficiency is a comparison of other high performance occupations with neurosurgeons, an attempt to understand the minimal number of cases required for proficiency during both residency training and after, and the effect of human factors.

Clinical Competence↗
PubMed2001

Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.

Amelogenesis Imperfecta↗
PubMed2001

Myc potentiates apoptosis by stimulating Bax activity at the mitochondria.

The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. During apoptosis, Bax translocation to the mitochondria occurs in the presence or absence of Myc expression. Moreover, Bax mRNA and protein expression remain unchanged in the presence or absence of Myc. However, in the absence of Myc, Bax is not activated and cytochrome c is not released into the cytoplasm. Reintroduction of Myc into the c-myc null cells restores Bax activation, cytochrome c release, and inhibition of apoptosis by Bcl-2. These results demonstrate a role for Myc in the regulation of Bax activation during apoptosis. Moreover, apoptosis that can be triggered in the absence of Myc provides evidence that signaling pathways exist which circumvent Bax activation and cytochrome c release to trigger caspase activation. Thus, Myc increases the cellular competence to die by enhancing disparate apoptotic signals at a common mitochondrial amplification step involving Bax activation and cytochrome c release.

Animals↗
PubMed2001

Assembly strategies and GTPase regulation of the eukaryotic and Escherichia coil translocons.

The translocation of most proteins across the endoplasmic reticulum or bacterial inner membrane occurs through an aqueous pore that spans the membrane. Substrates that are translocated co-translationally across the membrane are directed to the translocation pore via an interaction between the cytosolic signal recognition particle and its membrane-bound receptor. Together the translocation pore and the receptor are referred to as a translocon. By studying the biogenesis of the translocon a number of alternate targeting and membrane-integration pathways have been discovered that operate independently of the signal recognition particle (SRP) pathway. The novel assembly strategies of the translocon and the ways in which these components interact to ensure the fidelity and unidirectionality of the targeting and translocation process are reviewed here.

Animals↗
PubMed2000

Nucleotide-dependent binding of the GTPase domain of the signal recognition particle receptor beta-subunit to the alpha-subunit.

The signal recognition particle (SRP) receptor (SR) is a heterodimer of two polypeptides (SRalpha and SRbeta) that each contain a GTP-binding domain. The GTP-binding domain in the peripheral membrane SRalpha subunit has a well defined role in regulating targeting of SRP-ribosome-nascent chain complexes to the translocon. The only well established function for the transmembrane SRbeta subunit is anchoring SRalpha on the endoplasmic reticulum membrane. Deletion of the amino-terminal transmembrane domain of SRbeta did not affect receptor dimerization, but revealed a cryptic translocation signal that overlaps the GTPase domain. We demonstrate that the domain of SRalpha that binds SRbeta does so by binding directly to the nucleotide-bound form of the GTPase domain of SRbeta. An SRbeta mutant containing an amino acid substitution that allows the GTPase domain to bind XTP dimerized with SRalpha most efficiently in the presence of XTP or XDP, but not ATP. Our results suggest an additional level of regulation of SRP receptor function based on regulated dissociation of the receptor subunits.

Amino Acid Sequence↗
PubMed1999

Identification of the endoplasmic reticulum targeting signal in vesicle-associated membrane proteins.

The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane.

Adenosine Triphosphate↗
PubMed1999

A site-specific, membrane-dependent cleavage event defines the membrane binding domain of FtsY.

Targeting of many polytopic proteins to the inner membrane of prokaryotes occurs via an essential signal recognition particle-like pathway. Unlike the general secretory pathway, the proteins involved in this pathway and their activities appear in many respects to mirror closely those of their eukaryotic homologues. However, the Escherichia coli signal recognition particle receptor, FtsY, differs significantly at the amino terminus from the eukaryote homologue alpha-subunit of the signal recognition particle receptor. In addition, there is no prokaryote homologue of the transmembrane beta-subunit of the receptor. Therefore, FtsY must assemble on the membrane in a unique manner. Using assays designed to accurately discriminate membrane-bound proteins from aggregated material, we found that in contrast to a previous report, only amino acids 1-284 of FtsY are necessary and sufficient for membrane assembly. These amino acids together constitute a bona fide membrane binding domain that includes both the regions originally designated A and N based on sequence comparisons. Furthermore, we found that a membrane-bound factor mediates specific cleavage of some membrane-bound FtsY molecules between the N and G regions previously believed to be functionally linked to generate a novel membrane-bound isoform composed of only the AN domain.

Bacterial Proteins↗
PubMed1999

Negatively charged residues in the IgM stop-transfer effector sequence regulate transmembrane polypeptide integration.

A non-hydrophobic sequence that contributes to the biogenesis of a transmembrane protein is termed a stop-transfer effector (STE). To examine the mechanism of STE-mediated stop-transfer, a series of fusion proteins were constructed containing variants of a putative STE from murine IgM fused to an otherwise translocated hydrophobic sequence. Unexpectedly, the fraction of molecules adopting transmembrane topology was insensitive to many amino acid substitutions within the STE sequence but varied directly with the number of negative charges. Furthermore, when present at the amino terminus of a reporter, mutants were observed that adopted type I (amino terminus lumenal) and type II (amino terminus cytoplasmic) transmembrane topologies, demonstrating that the STE sequence can be located at either side of the endoplasmic reticulum membrane. Our results suggest that recognition of a broad structural feature formed primarily by negatively charged residues within the STE halts translocation and triggers membrane integration, even when the negative charges end up on the cytoplasmic side of the membrane. Since functional STE sequences photocross-link to two membrane proteins not previously identified at the translocon, these unique proteins are presumably involved in recognizing STE sequences and/or facilitating STE function.

Amino Acid Sequence↗
PubMed1999

Regulation of acidification and apoptosis by SHP-1 and Bcl-2.

Recruitment of the SH2 domain containing cytoplasmic protein-tyrosine phosphatase SHP-1 to the membrane by somatostatin (SST) is an early event in its antiproliferative signaling that induces intracellular acidification-dependent apoptosis in breast cancer cells. Fas ligation also induces acidification-dependent apoptosis in a manner requiring the presence of SHP-1 at the membrane. Moreover, we have recently reported that SHP-1 is required not only for acidification, but also for apoptotic events that follow acidification (Thangaraju, M., Sharma, K., Liu, D., Shen, S. H., and Srikant, C. B. (1999) Cancer Res. 59, 1649-1654). Here we show that ectopically expressed SHP-1 was predominantly membrane-associated and amplified the cytotoxic signaling initiated upon SST receptor activation and Fas ligation. The catalytically inactive mutant of SHP-1 (SHP-1C455S) abolished the ability of the SST agonists to signal apoptosis by preventing the recruitment of wild type SHP-1 to the membrane. Overexpression of the anti-apoptotic protein Bcl-2 in MCF-7 cells inhibited SST-induced apoptosis upstream of acidification by inhibiting p53-dependent induction of Bax as well as by raising the resting pH(i) and attenuating SST-induced decrease in pH(i). By contrast, Bcl-2 failed to prevent apoptosis triggered by direct acidification. These data demonstrate that (i) membrane-associated SHP-1 is required for receptor-mediated cytotoxic signaling that causes intracellular acidification and apoptosis, and (ii) Bcl-2 acts distal to SHP-1 and p53 to prevent SST-induced acidification but cannot inhibit the apoptotic events that ensue intracellular acidification.

Amiloride↗
PubMed1999

Bcl-2 targeted to the endoplasmic reticulum can inhibit apoptosis induced by Myc but not etoposide in Rat-1 fibroblasts.

Bcl-2 is a key inhibitor of a broad range of apoptotic pathways, yet neither the mechanism of action nor the role of Bcl-2 subcellular localization are well understood. The subcellular localization of Bcl-2 includes the mitochondrial membrane as well as the contiguous membrane of the endoplasmic reticulum and nuclear envelope. Most studies suggest that the ability of Bcl-2 to confer cell survival is dependent upon its localization to the mitochondria. In this manuscript, we show that Bcl-2 targeted to the endoplasmic reticulum can inhibit Myc-, but not etoposide-induced apoptosis in the Rat-1 fibroblast cell line. By contrast, wild type Bcl-2 can inhibit apoptosis triggered by either death agonist. We further show both Myc and etoposide trigger disruption of mitochondrial membrane potential (MMP) and induce poly-ADP ribose polymerase (PARP) cleavage, but release of calcium was not evident. Bcl-2 abrogates apoptosis at or upstream of MMP depletion showing that Bcl-2 does not have to reside at the mitochondria to prevent apoptosis. These results further elucidate the biochemical events associated with Myc- and etoposide-induced apoptosis and significantly advance our understanding of Bcl-2 function.

Animals↗