Biomedical subjects
Publications and source records attributed to D W Andrews.
This article focuses on developments in treatment options for pituitary adenomas. We review the literature on the advances in diagnosis and treatment modalities, including medical and surgical approaches. We also discuss conventional radiation and the recently proposed genetic treatments for pituitary tumors.
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The purpose of this study was to demonstrate improved cranial nerve tolerance following fractionated stereotactic radiotherapy for large acoustic neuromas, defined as tumours with pons-petrous distance (A) > 1 cm and midporous transverse diameter (A + Y) > 2 cm. Of 28 patients with acoustic neuromas treated with fractionated stereotactic radiotherapy, 19 had large tumours at high risk for radiosurgery-induced cranial neuropathy. Six patients received 36 Gy in six, weekly, fractions and 13 patients received 30 Gy in six, weekly, fractions. 15 patients had evaluable trigeminal nerve function and 16 had evaluable facial nerve function. At a median follow-up of 4.5 years, tumour shrinkage was seen in 10 patients and tumour size was stable in nine. None of the patients developed any evidence of transient, or permanent, trigeminal or facial neuropathy at any time during their follow-up period. Fractionated stereotactic radiotherapy may offer a superior therapeutic ratio to single fraction stereotactic radiosurgery in the management of large acoustic neuromas, as evidenced by the absence of post-treatment trigeminal and facial neuropathy.
A 71-year-old male presented with a large pituitary adenoma with superior extension into the optic chiasm and suprasellar cistern. He was treated with stereotactic radiosurgery to a dose of 16 Gy. Approximately 1 h after radiosurgery he developed fever; his temperature peaked at 105.1 degrees F and normalized about 20 h later. This case demonstrates that acute hyperthermia is a potential complication following high dose stereotactic radiosurgery for large pituitary tumours.
To examine the early cellular changes that accompany transformation, middle-T antigen (the transforming protein of polyomavirus), was expressed from an inducible promoter in Rat2 fibroblasts and in normal diploid human fibroblast (MRC5) cells using a replication defective adenovirus vector. The earliest detectable expression of middle-T antigen correlated closely with the initial changes in cellular morphology. At this time, expression of middle-T antigen resulted in the redistribution of shc and src to a brefeldin A resistant perinuclear compartment coincident with the formation of kinase active complexes containing middle-T antigen, shc and src. The first detectable changes directly related to altered morphology was reorganization of actin filaments, and the condensation of tubular endosomes in the perinuclear region of the cell. These results suggest a potential role for perinuclear localized mT molecules in some of the morphologic changes associated with the initial phase of cellular transformation.
Interleukin-16, a proinflammatory cytokine produced in CD8(+) lymphocytes, is synthesized as a precursor protein (pro-IL-16). It is postulated that the C-terminal region of pro-IL-16 is cleaved, releasing bioactive IL-16. To characterize IL-16 cleavage, we transfected COS cells with a cDNA encoding a approximately 50-kDa form of pro-IL-16. Transfected COS cells released a approximately 20-kDa IL-16 cleavage product shown to consist of the 121 C-terminal residues of pro-IL-16 by immunoblotting and amino acid sequencing. Cleaved IL-16, but not pro-IL-16, exhibited lymphocyte chemoattractant activity. A C-terminal approximately 20-kDa IL-16 polypeptide was also released when pro-IL-16 was treated with concanavalin A-stimulated CD8(+) lymphocyte lysate. Cleavage occurred after an Asp, suggesting involvement of a caspase (interleukin-1beta-converting enzyme/CED-3) family protease. Using recombinant caspases and granzyme B, we determined that pro-IL-16 cleavage is mediated only by caspase-3. Relevance to pro-IL-16 processing in primary lymphocytes was supported by identifying the p20 subunit of activated caspase-3 in stimulated CD8(+) lymphocytes and by inhibition of CD8(+) lymphocyte lysate-mediated cleavage with Ac-DEVD-CHO. Pro-IL-16 is a substrate for caspase-3, and cleavage by this enzyme releases biologically active IL-16 from its inactive precursor.
Networks of interstitial cells of Cajal embedded in the musculature of the gastrointestinal tract are involved in the generation of electrical pacemaker activity for gastrointestinal motility. This pacemaker activity manifests itself as rhythmic slow waves in membrane potential, and controls the frequency and propagation characteristics of gut contractile activity. Mice that lack a functional Kit receptor fail to develop the network of interstitial cells of Cajal associated with Auerbach's plexus in the mouse small intestine and do not generate slow wave activity. These cells could provide an essential component of slow wave activity (for example, a biochemical trigger that would be transferred to smooth muscle cells), or provide an actual pacemaker current that could initiate slow waves. Here we provide direct evidence that a single cell, identified as an interstitial cell of Cajal by light microscopy, electron microscopy and expression of Kit mRNA, generates spontaneous contractions and a rhythmic inward current that is insensitive to L-type calcium channel blockers. Identification of the pacemaker of gut motility will aid in the elucidation of the pathophysiology of intestinal motor disorders, and provide a target cell for pharmacological treatment.
The 5' untranslated region of an RNA molecule is thought to play an important role in the regulation of translation. Following a recent report that a single nucleotide is sufficient to act in this role in the unicellular organism Giardia, we show that this is also the case for a mammalian in vitro system. These results also demonstrate that an RNA can initiate translation from a start codon where an ideal translational consensus sequence is impossible.
Subcellular localization of proteins with carboxyl-terminal insertion sequences requires the molecule be both targeted to and integrated into the correct membrane. The mechanism of membrane integration of cytochrome b5 has been shown to be promiscuous, spontaneous, nonsaturable, and independent of membrane proteins. Thus endoplasmic reticulum localization for cytochrome b5 depends primarily on accurate targeting to the appropriate membrane. Here direct comparison of this mechanism with that of three other proteins integrated into membranes via carboxyl-terminal insertion sequences [vesicle-associated membrane protein 1(Vamp1), polyomavirus middle-T antigen, and Bcl-2] revealed that, unlike cytochrome b5, membrane selectivity for these molecules is conferred at least in part by the mechanisms of membrane integration. Bcl-2 membrane integration was similar to that of cytochrome b5 except that insertion into lipid vesicles was inefficient. Unlike cytochrome b5 and Bcl-2, Vamp1 binding to canine pancreatic microsomes was saturable, ATP-dependent, and abolished by mild trypsin treatment of microsomes. Surprisingly, although the insertion sequence of polyomavirus middle-T antigen was sufficient to mediate electrostatic binding to membranes, binding did not lead to integration into the bilayer. Together these results demonstrate that there are at least two different mechanisms for correct membrane integration of proteins with insertion sequences, one mediated primarily by targeting and one relying on factors in the target membrane to mediate selective integration. Our results also demonstrate that, contrary to expectation, hydrophobicity is not sufficient for insertion sequence-mediated membrane integration. We suggest that the structure of the insertion sequence determines whether or not specific membrane-bound receptor proteins are required for membrane integration.
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A new type of fish antifreeze protein, designated here type IV, has been isolated from the longhorn sculpin, Myoxocephalus octodecimspinosis. Sequence analysis of the protein (LS-12) reveals that it contains 108 amino acids, is blocked at the N-terminus by a pyroglutamyl group and has a high (17%) content of glutamine; it is thus completely unrelated to the earlier described types I, II and III fish antifreeze proteins. Circular dichroism spectra and conformational analysis based on the sequence data indicate that LS-12 has a high helix content and probably folds as a four-helix bundle. LS-12 shows sequence similarity to certain plasma apolipoproteins known to have helix bundle structures, suggesting the possibility that LS-12 may have arisen by recruitment and mutation of a plasma apolipoprotein.
External beam irradiation of malignant astrocytoma often provides temporary local tumor control, but dose is limited by potential toxicity to normal brain. Fractionated stereotactic radiotherapy (SRT) provides additional radiation to the tumor with less dose deposition in adjacent normal brain. We administered a potential radiosensitizer, cis-platinum (CDDP), to optimize the therapeutic index. CDDP (40 mg/m2) was given weekly, with SRT once or twice weekly, to 20 patients. One had a partial response, 11 stable disease, and eight progressed despite therapy. Acute toxicities were manageable. Five patients required surgery for tumor progression or radiation necrosis. Median response duration was 18.5 weeks and median survival was 55 weeks. SRT combined with CDDP is safe, with durable responses in some patients. Further investigations to determine optimal SRT and CDDP doses and scheduling are justified.
This review focuses on recent developments in the treatment of pituitary adenomas. Current treatments include both transcranial and transsphenoidal surgery, the medical treatment specifically for endocrinopathies, and finally, radiation treatments including conventional radiation as well as stereotactic radiosurgery. All these modalities are reviewed.
OBJECTIVE: To explore the presence of MDR-1 drug resistance in human glioma utilizing a Single Photon Emission Computerized Tomography (SPECT) imaging agent, sesta-MIBI, and testing cases interpreted as positive for drug resistance with molecular characterization of subsequent tissue biopsy, including RNA, Northern blot analysis, and polymerase chain reaction, and in situ hybridization. METHODS: Six patients that carried a diagnosis of biopsy-proven glioma underwent dual isotope SPECT imaging with thallium 201 and technetium sesta-MIBI. All six patients underwent subsequent surgical reexcision of their tumors, and tissue was immediately flash frozen for further analysis. PolyA RNA was isolated and subsequently analyzed by both Northern blot analysis and RT-PCR utilizing an MDR-1 probe (ATCC) and MDR-1 primers. RESULTS: Four patients with SPECT concordance yielded tumors without MDR-1 expression whereas two patients with SPECT discordance yielded tumors with MDR-1 gene expression. In one discordant case we subsequently performed RT-PCR in situ amplification/ hybridisation and immunohistochemistry with, respectively, an MDR-1 sense primer with Biotin labeled probes and an MDR-1 monoclonal antibody, and both analyses revealed MDR-1 expression in tumor cells. CONCLUSION: These data support the use of SPECT technetium sesta-MIBI to evaluate the presence of MDR-1 gene expression in gliomas.
OBJECTIVE: We describe our initial experience with fractionated stereotactic radiotherapy (SRT) for the treatment of 19 patients with secretory and nonsecretory pituitary macroadenomas. The primary endpoints of local control and the documentation of any MRI T2-weighted changes in contiguous brain tissues are discussed. METHODS: Between 1/95 and 1/97, 19 patients were planned with the X-Knife 3-D planning system (Radionics, Burlington, Mass., USA) and received a median dose of 46 Gy in daily 2.0-Gy fractions. Treatments were delivered stereotactically with a dedicated 600SR linear accelerator (Varian Corporation, Palo Alto, Calif., USA). Immobilization was achieved with the Gill-Thomas-Cosman relocatable frame. The mean tumor size was 2.24 cm. The mean prescription isodose was 87%. The mean age was 53 years (10 male, 9 female). The mean follow-up time was 10 months (range 1-24 months). The mean optic chiasm and brain stem doses were calculated at 38 and 13 Gy, respectively. All patients were evaluated with pre- and postgadolinum-enhanced MRI scans and Humphrey visual field tests. RESULTS: In the posttreatment period, local control (absence of tumor progression) has been achieved in all of the patients. The treatment was well tolerated in all patients. No acute complications, no visual changes and no T2-weighted MRI or proton density changes were documented in any of the 19 patients. CONCLUSION: These preliminary results suggest that SRT compares favorably with conventional radiotherapy in achieving local control. The doses to the brainstem and the temporal lobes are significantly decreased and at early follow-up no white matter changes are seen on MRI after SRT. The true frequency of grade 1-4 changes are likely underestimated as similar changes often occur in association with tumor edema or after surgery. Given the uncertain neurocognitive significance of the white matter changes associated with treating these benign tumors by conventional radiotherapy, we are currently treating all pituitary adenomas with fractionated SRT to reduce the potential sequelae.
Bax, a member of the Bcl-2 family of proteins, has been shown to accelerate apoptosis induced by growth factor withdrawal, gamma-irradiation, and the chemotherapeutic agent, etoposide. The mechanism by which Bax promotes apoptosis is poorly understood. Bax forms homodimers which have been suggested to act as accelerators or inducers of cell death. However, the requirement for homodimerization of Bax to promote cell death remains unclear. We performed site-directed mutagenesis of the BH1, BH2, and BH3 in Bax to determine the regions of Bax required for homodimerization and to define the role of Bax homodimers in cell death induced by chemotherapy drugs. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 (G108) in BH1, tryptophan 158 (W158) in BH2, and glycine 67 and aspartic acid 68 (GD67-68) in BH3 as well as deletion of the most conserved amino acids in BH1 (Delta102-112) and BH2 (Delta151-159) and deletion of BH3 (Delta63-71) maintained their ability to accelerate chemotherapy-induced cell death. Immunoprecipitation studies revealed that Bax with deletions in BH1 and BH2 still associated with wild-type Bax while deletion of BH3 disrupted Bax homodimerization. These results demonstrate that Bax does not require the conserved regions of homology, BH1, BH2, or BH3, to accelerate chemotherapy-induced cell death. Furthermore, our results established BH3 as a region required for Bax homodimerization in mammalian cells and demonstrate that monomeric forms of Bax are active in accelerating cell death induced by chemotherapy agents.
Human Bcl-2 is located in multiple intracellular membranes when expressed in MDCK and Rat-1/myc cells. We restricted expression to the endoplasmic reticulum or mitochondria by exchanging the Bcl-2 carboxy-terminal insertion sequence for an equivalent sequence from cytochrome b5 or ActA, respectively. MDCK cells are protected from serum deprivation-induced apoptosis by both wild-type Bcl-2 and the mutant targeted to mitochondria but not by the mutant targeted to endoplasmic reticulum. In contrast, when expressed in Rat-1/myc cells, the Bcl-2 mutant located at the endoplasmic reticulum is more effective than that targeted to mitochondria. In MDCK cells both mutants bind Bax as effectively as wild-type, demonstrating that Bax binding is not sufficient to prevent apoptosis.
The herpes simplex virus (HSV) ICP47 protein inhibits the MHC class I antigen presentation pathway by inhibiting the transporter associated with antigen presentation (TAP) which translocates peptides across the endoplasmic reticulum membrane. At present, ICP47 is the only inhibitor of TAP. Here, we show that ICP47 produced in bacteria can block human, but not mouse, TAP, and that heat denaturation of ICP47 has no effect on its ability to block TAP. ICP47 inhibited peptide binding to TAP without affecting ATP binding, consistent with previous observations that the peptide binding and ATP binding sites of TAP are distinct. ICP47 bound to TAP with a higher affinity (KD approximately 5 x 10(-8) M) than did peptides, and ICP47 did not dissociate from TAP. ICP47 was not transported by TAP and remained sensitive to proteases added from the cytosolic surface of the membrane. Peptides acted as competitive inhibitors of ICP47 binding to TAP, and this inhibition required a 100- to 1000-fold molar excess of peptide. These results demonstrate that ICP47 binds to a site which includes the peptide binding domain of TAP and remains bound to this site in a stable fashion.