Biomedical subjects

R A Fenstermaker

Publications and source records attributed to R A Fenstermaker.

At least 19 recordsLinked to original sources

PubMed2001

2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) in a nude rat glioma model: implications for photodynamic therapy.

BACKGROUND AND OBJECTIVE: In this study, we evaluated 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-alpha (HPPH or Photochlor) as a photosensitizer for the treatment of malignant gliomas by photodynamic therapy (PDT). STUDY DESIGN/MATERIALS AND METHODS: We performed in vivo reflection spectroscopy in athymic rats to measure the attenuation of light in normal brain tissue. We also studied HPPH pharmacokinetics and PDT effects in nude rats with brain tumors derived from stereotactically implanted U87 human glioma cells. Rats implanted with tumors were sacrificed at designated time points to determine the pharmacokinetics of HPPH in serum, tumor, normal brain, and brain adjacent to tumor (BAT). HPPH concentrations in normal brain, BAT and tumor were determined using fluorescence spectroscopy. Twenty-four hours after intravenous injection of HPPH, we administered interstitial PDT treatment at a wavelength of 665 nm. Light was given in doses of 3.5, 7.5 or 15 J/cm at the tumor site and at a rate of 50 mW/cm. RESULTS: In vivo spectroscopy of normal brain tissue showed that the attenuation depth of 665 nm light is approximately 30% greater than that of 630 nm light used to activate Photofrin, which is currently being evaluated for PDT as an adjuvant to surgery for malignant gliomas. The t1/2 of disappearance of drug from serum and tumor was 25 and 30 hours, respectively. CONCLUSION: Twenty-four hours after injection of 0.5 mg/kg HPPH, tumor-to-brain drug ratios ranged from 5:1 to 15:1. Enhanced survival was observed in each of the HPPH/PDT-treated animal groups. These data suggest that HPPH may be a useful adjuvant for the treatment of malignant gliomas.

Animals↗

PubMed2001

Evaluation of systemically administered radiolabeled epidermal growth factor as a brain tumor targeting agent.

We have previously reported a method for labeling epidermal growth factor (EGF) with technetium-99m and have shown that 99mTc-EGF localized in EGF receptor (R) positive intracerebral C6EGFR rat gliomas following intratumoral (i.t.) injection of the radioligand. In the present study, we have evaluated the potential use of 99mTc-EGF as a tumor targeting agent after systemic administration to Fischer rats bearing intracerebral implants of C6EGFRgliomas. Radiolocalization was determined following intravenous (i.v.) or intracarotid (i.c.) injection with or without hyperosmotic mannitol induced disruption of the blood-brain barrier (BBB-D). As determined by gamma-scintillation counting, 4 h after i.c. injection of 99mTc-EGF, 0.34% of the injected dose per gram (% ID/g) was localized in C6EGFR tumors. which expressed 10(5)-10(6) EGFR sites per cell, compared to 0.07% ID/g in animals bearing C6 wildtype gliomas, which do not express EGFR. The corresponding tumor to brain ratios were 5.6 and 1.6, respectively. Tumors could be visualized by external gamma-scintigraphy in rats bearing C6EGFR but not C6 wildtype gliomas, thereby establishing that radiolocalization was dependent upon receptor expression. Intracarotid administration of 99mTc-EGF significantly increased tumor uptake compared to i.v. injection (0.34 vs 0.14% ID/g, p < 0.04). BBB-D disruption, followed by i.c. injection of 99mTc-EGF, however, did not significantly enhance tumor uptake compared to i.c. injection without BBB-D (0.45% vs 0.34% ID/g, p > 0.1). The uptake of 99mTc-EGF was approximately 4-9% ID/g in the liver and 12-20% ID/g in the kidneys after i.c. or i.v. administration. External gamma-scintigraphy of regions of interest over the liver and kidneys revealed that approximately 70-80% of the whole body radioactivity accumulated in these organs, and only 0.47-0.83% in the tumor following i.v. or i.c. administration of 99m9Tc-EGF. Our study has demonstrated that EGF can be used as a specific targeting agent for EGFR (+) rat brain tumors. However, it is unlikely that systemic injection of EGF-based bioconjugates can deliver sufficient amounts of the ligand to brain tumors for therapeutic purposes and direct delivery by means of either intratumoral injection or a variant of it such as convection enhanced delivery will be required.

Animals↗

PubMed2000

Deletion and tandem duplication of exons 2 - 7 in the epidermal growth factor receptor gene of a human malignant glioma.

The epidermal growth factor receptor (EGFR) gene is frequently amplified and rearranged in malignant gliomas with expression of oncogenic deletion mutants (DM). The most common mutant EGFRvIII, which contains a deletion of exons 2 - 7, is constitutively autophosphorylated and inefficiently downregulated. Other less common EGFR mRNA species in gliomas contain tandem duplication of exons, which encode the tyrosine kinase (TK) and calcium mediated receptor internalization (CAIN) domains of the molecule. We examined a panel of human malignant gliomas and found one tumor that expressed four related EGFR proteins, including 125-, 140-, 170-, and 180-kDa species. This tumor also contained four EGFR-related mRNA species, including both wild type EGFR and EGFRvIII transcripts. A third transcript contained a deletion of exons 2 - 7 and 12 - 13 corresponding to the 125-kDa protein. A fourth transcript contained an in-frame, tandem duplication of exons 2 - 7 (EGFR.TDM/2 - 7). The 180-kDa, tandem duplication mutant (TDM) exhibited enhanced basal phosphorylation and impaired downregulation. In contrast to the 140-kDa EGFRvIII; however, phosphorylation of the 180-kDa EGFR.TDM/2 - 7 was strongly induced by ligand. Expression of both deletion and tandem duplication mutants in the same tumor suggests that the mechanisms responsible for DM and TDM formation might be closely related. Oncogene (2000) 19, 4542 - 4548.

Central Nervous System Neoplasms↗

PubMed2000

Oncogenic epidermal growth factor receptor mutants with tandem duplication: gene structure and effects on receptor function.

A number of epidermal growth factor receptor (EGFR) deletion mutants have been identified in gliomas, in which the EGFR gene is frequently amplified and rearranged. We have previously characterized the structure of a gene in A-172 human glioma cells that encodes a 190-kDa EGFR mutant with tandem duplication of the tyrosine kinase (TK) and calcium-mediated internalization (CAIN) domains. Here we describe a 185-kDa tandem duplication mutant (TDM) that is expressed in KE and A-1235 glioma cells, along with certain functional characteristics of the mutants. The corresponding transcripts in KE and A-1235 cells contain 1053 additional nucleotides representing an in-frame duplication of exons 18 through 25 which encode the entire TK region and a portion of the CAIN domain. As with duplication of the entire TK/CAIN region (exons 18-26) in A-172 cells, duplication of exons 18-25 is associated with a specific genomic rearrangement between flanking introns. Involved introns contain homology to recombination signal sequence (RSS) heptamers present in the V(D)J region of the T lymphocyte receptor gene. In defined medium, both oncogenic TDM are constitutively autophosphorylated and inefficiently downregulated. High-affinity binding is reduced in EGFR.TDM/18-26, although the t1/2 of receptor internalization is not prolonged.

Animals↗

PubMed2000

The impact of age and sex on the incidence of glial tumors in New York state from 1976 to 1995.

OBJECT: In this study the authors describe secular trends in the incidence of three glial tumors--glioblastoma multiforme (GBM), astrocytoma not otherwise specified (ANOS), and anaplastic astrocytoma (AA)--in New York state from 1976 through 1995. They also describe the effect of age and sex on the relative risk (RR) for these tumors, specifically GBM. METHODS: Crude, age-, and sex-specific incidence rates were calculated for each tumor type from 1976 to 1995 by using data from the New York State Cancer Registry. Age-adjusted incidence rates were calculated by the direct standardization procedure, in which the 1970 United States Census Population Standard Million is used. The RR of GBM for the female population was calculated and plotted. Statistical comparisons were made using Pearson's correlation coefficient and regression analysis with the coefficient of variation. CONCLUSIONS: The age-adjusted incidence of these three glial tumors increased during the study period. Increases in age-specific incidence of GBM were primarily limited to patients 60 years of age or older. The reasons for these increases cannot be fully explained with the data. Those in the female population had a lower risk of developing these tumors than those in the male. For GBM, the protective effect of sex was first evident at the approximate age of menarche, was greatest at the approximate age of menopause, and decreased in postmenopausal age strata. The overall protective effect of female sex and the described trend in RR for GBM in the female population suggests that sex hormones and/or genetic differences between males and females may play a role in the pathogenesis of this tumor.

Adult↗

PubMed1999

Synergistic cytotoxicity, apoptosis and protein-linked DNA breakage by etoposide and camptothecin in human U87 glioma cells: dependence on tyrosine phosphorylation.

In this study, simultaneous administration of certain inhibitors of topoisomerase I and topoisomerase II produced synergistic cytotoxicity in a series of human glioma cell lines. Camptothecin (CPT) and etoposide (VP-16) produced combination indices (CI) <1.0 in all glioma cell lines tested, including those that were relatively resistant to the two topoisomerase inhibitors individually. In contrast, CPT and VP-16 produced additive cytotoxicity in HT-29 and SW-620 colon carcinoma cell lines. To explore the molecular basis for synergy in glioma cells, we focused on one glioma cell line (U87) in which even sub-cytotoxic doses of CPT potentiated the action of VP-16. Except for genistein (a topo II agent with tyrosine kinase inhibitory function), all topo II inhibitors tested (doxorubicin, ellipticine, and m-AMSA) were synergistic with CPT. While CPT and VP-16 produced cytotoxicity and protein-linked DNA breaks (PLDB) that were supra-additive in U87 glioma cells, CPT and genistein produced additive results. Pretreatment of U87 cells with the tyrosine kinase inhibitor tyrphostin-A23 or the tyrosine phosphatase activator O-phospho-L-tyrosine (OPLT) reduced combination PLDB from synergistic to additive levels, but had no effect on the formation of PLDB induced by either CPT or VP-16 alone. CPT and VP-16 also produced a synergistic accumulation of sub-G0 (apoptotic) cells which was blocked by tyrphostin-A23. No significant increase in topoisomerase protein levels could be detected in response to combination treatment. Thus, synergistic effects between topoisomerase I and topoisomerase II inhibitors in U87 glioma cells may depend upon phosphorylation of cellular proteins other than the topoisomerases themselves.

Brain Neoplasms↗

PubMed1998

Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin.

Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF alpha-and beta-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK-->EEE), whereas an RKK-->SSS mutant also exhibited a modest loss in affinity for beta-receptors. Replacements with serine at either Arg160 (RKK-->SKK) or at all three positions (RKK-->SSS) had little effect on binding to alpha-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK-->EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK-->EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK-->EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of beta-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties.

Animals↗

PubMed1998

Tandem duplication of the epidermal growth factor receptor tyrosine kinase and calcium internalization domains in A-172 glioma cells.

Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene occur frequently in malignant gliomas. Rearrangement may also lead to the expression of potentially oncogenic EGFR deletion mutants. Data presented here indicate the existence of a 190 kDa mutant form of the EGFR in A-172 glioma cells that is substantially different from the deletion mutants characterized previously. The EGFR-like protein is expressed along with the 170 kDa wild type EGFR. It is detectable with antibodies to both extracellular and intracellular regions of the EGFR, but is not crossreactive with other HER-family members. The wild type and mutant receptors undergo phosphorylation in response to treatment with TGFalpha and are associated with expression of both 10.5 kb and 11.5 kb EGFR-related transcripts. Combined reverse transcription-polymerase chain reaction (RT-PCR) identifies a unique transcript in A-172 cells that encodes an in-frame, tandem duplication of both tyrosine kinase and calcium internalization (TK/CAIN) domains (exons 18 through 26). The duplication of these domains is associated with a specific genomic rearrangement between potential v-myb and c-myb consensus binding sites within introns 26 and 17 of the EGFR gene resulting in the formation of a chimeric intron.

Antigens, Neoplasm↗

PubMed1998

Intratumoral infusion of topotecan prolongs survival in the nude rat intracranial U87 human glioma model.

Topotecan is a topoisomerase (topo) I inhibitor with promising activity in preclinical studies. We hypothesized that low-dose intratumoral delivery of topotecan would be highly effective for gliomas. Human glioma cell lines (U87, U138 and U373) displayed different sensitivities to topotecan (IC50 range: 0.037 microM to 0.280 microM) in cell culture. The most resistant of the glioma cell lines (U87) was implanted stereotactically into the brains of nude rats. Twelve days later, at which time tumor diameter measured 2 to 2.5 mm, animals were randomized to three groups: group I, intratumoral topotecan infused via osmotic pump (n = 12); group II, intratumoral saline infusion (n = 7); and group III, no treatment (n = 10). Animals were sacrificed when signs of deterioration developed, or at 60 days. Animals in group I had a mean survival time (MST) of > 55 days (range = 40-60); whereas, those in groups II and III had MST of 26.1 (range = 21-31) and 26.5 (range = 20-30) days, respectively. The differences in survival between group I and each of the other groups were statistically significant (p < 0.0001; Logrank Mantel-Cox). None of the animals that survived 60 days had histological evidence of residual tumor at sacrifice. Measurement of topotecan levels in normal brain revealed cytotoxic concentrations up to 4.5 mm from the site of infusion. This study demonstrates that intratumoral topotecan delivered via an osmotic pump prolongs survival in the U87 human glioma model.

Animals↗

PubMed1998

Correlation of intraluminal thrombosis in brain tumor vessels with postoperative thrombotic complications: a preliminary report.

OBJECT: Thrombotic complications (deep vein thrombosis and/or pulmonary embolization [DVT/PE]) occur in 18 to 50% of patients harboring brain tumors who undergo neurosurgical procedures. Such patients are at risk for DVT/PE because of immobility, paresis, hypovolemia, and lengthy surgery. The present study was undertaken to see whether tumor patients at highest risk for DVT/PE could be identified so that augmentation of prophylactic measures might be used to reduce the incidence of thrombotic complications. METHODS: The authors conducted a retrospective analysis of 488 patients enrolled in their brain tumor registries between 1988 and 1995, identifying 57 patients (12%) with recorded symptomatic DVT, PE, or both postoperatively. In 24 of these 57 cases histological specimens were retrievable for review, allowing an in-depth analysis. Forty-five patients were lost to follow-up review, and the remaining 386 patients had no record of systemic thrombosis. Slides of pathological specimens were retrievable in 50 cases in which there was no DVT/PE. From these 50 cases, 25 were selected at random to represent the control group by a blinded observer. Seventeen (71%) of the 24 brain tumor specimens obtained in patients with DVT/PE stained positively for intraluminal thrombosis (ILT) after hematoxylin and eosin had been applied. The odds ratio associated with the presence of ILT was 17.8, with a confidence interval ranging from 4 to 79.3. No evidence of ILT was found in 22 patients (88%) within the control group (p < 0.0001, Fisher's exact test). Other factors that may predispose patients with brain tumors to DVT/PE-limb paresis, extent of tumor removal, and duration of the surgery-were also analyzed and found not to be statistically significant. Therefore, these factors were not the basis for differences seen between the study and control groups. CONCLUSIONS: These preliminary observations suggest that the presence of ILT within malignant glioma or glioblastoma tumor vessels may represent a marker of tumor-induced hypercoagulability.

Adult↗

PubMed1996

Site-directed mutagenesis of the N-linked glycosylation site in platelet-derived growth factor B-chain results in diminished intracellular retention.

Pulse-chase analysis of human platelet-derived growth factor (PDGF) B-chain was conducted in stably transfected Chinese hamster ovary cells to determine precisely the kinetics of processing, intracellular trafficking and secretion. Newly synthesized 31 kDa monomers of the B-chain (p31) dimerized rapidly via disulfide bonds to a p54 species (t1/2 < 30 min). The p54 dimer was processed to a group of intracellular, cell surface (suramin-releasable) and secreted forms whose rates of appearance and disappearance from the cell were measured over a 48 h period. The newly synthesized p31 species was quantitatively converted to p27 by treatment with endoglycosidase H, consistent with efficient N-glycosylation at a site in the N-terminal propeptide region (Asn63-Met64-Thr65). Interruption of B-chain glycosylation by oligodeoxynucleotide-directed mutagenesis resulted in a significant increase in suramin-releasable forms at the cell surface (p34-38) and a concomitant decrease in accumulation of an intracellular p24 species. The glycosylation-defective mutant exhibited slight increases in receptor binding and mitogenic activity. Our results suggest that N-linked glycosylation of the B-chain is not important for formation of mitogenically active protein, but that it plays a role in early intracellular sorting and proteolytic processing events.

Animals↗

PubMed1995

CT-assisted percutaneous anterior cordotomy: technical note.

Percutaneous radiofrequency cordotomy remains a useful modality for managing selected patients with unremitting pain due to malignancy. Although the lateral C1-C2 method is widely used, certain clinical situations may call for a low anterior cervical approach via the disc space. The anterior method can be technically exacting due to the need for precision in the angle of approach at the point of disc space entry. Here we describe how CT-imaging can be used to simplify target localization during percutaneous anterior cervical cordotomy.

Cordotomy↗

PubMed1995

The effect of epidermal growth factor receptor (EGFR) expression on in vivo growth of rat C6 glioma cells.

The discovery of EGFR gene amplification in glioblastoma multiforme has prompted interest in experimental therapies to target the receptor on brain tumor cells. To develop an animal model for in vivo study of such strategies, we transfected C6 glioma cells with a plasmid containing the neomycin resistance gene and the human EGFR gene under the control of the glucocorticoid-inducible MMTV promoter. Following selection with G418, individual clones that expressed EGFR at high levels were selected. Kinetics of EGF binding fit a dual site model indicating the presence of both high (KA = 2.5 x 10(9) M-1) and low (KA = 3.3 x 10(7) M-1) affinity receptors. To assess growth in vivo, graded numbers of either wild-type or transfected cells were implanted into the brains of CD Fischer 344 rats. No differences in survival were observed between groups of animals injected with either wild-type or transfected cells at inocula of 10(3) or 10(4) respectively. In addition, one-third of animals (7/21) challenged with 10(5) or 10(6) transfected cells survived > 50 days compared to 0% of animals (0/12) challenged with 10(5) or 10(6) wild-type cells. Such an effect suggests greater immunogenicity of transfected cells, but only at the larger inocula. Since C6 glioma cells will grow in both outbred and inbred strains, our model should have a number of applications including the in vivo study of EGFR targeting for glioma therapy.

Animals↗

PubMed1994

Platelet-derived growth factor A-chain gene transcription is mediated by positive and negative regulatory regions in the promoter.

Platelet-derived growth factor (PDGF) is a disulphide-linked heterodimer of two polypeptide chains, the A and B chains, which are encoded by genes on separate chromosomes. The A-chain gene is transcribed in a number of transformed and non-transformed cell lines and is inducible by a wide variety of growth factors, cytokines and other mitogenic agonists. To localize DNA elements that mediate basal transcription in the promoter regulatory region of the A-chain gene, we have employed 5'-endpoint deletion mutagenesis and transient expression analysis in the renal epithelial cell line BSC-1 (African green monkey). Studies conducted in this cell line, which expresses high concentrations of PDGF A-chain mRNA, reveal a positive regulatory element (PRE) in a GC-rich stretch of the A-chain promoter between -82 and -40, relative to the transcription start site. Two discrete regions of the promoter were identified as negative regulatory elements (NREs), located between -1029 and -880 (NRE1) and between -1800 and -1029 (NRE2). The -1800 to -812 region, which contains both NREs, functions as a potent NRE when relocated in either orientation adjacent to the herpes simplex virus thymidine kinase promoter, reducing transcription activity by 60% in the positive orientation and 85% in the negative orientation. Comparison of BSC-1 cells and Saos-2 cells (human osteogenic sarcoma), which do not express significant quantities of PDGF A-chain mRNA or protein, indicates that basal transcription of the gene is determined by enhancer activity mediated by the GC-rich region rather than through de-repression of the upstream NREs. Electrophoretic gel mobility shift assays reveal a complex pattern of nuclear protein binding to the GC-rich PRE (-73 to -46). Competition studies conducted with mutant oligonucleotides that alternately disrupt consensus binding sites for Sp-1 or Egr-1 demonstrate a requirement for the presence of an Sp1-like core sequence (GGCGGG) but not Egr-1/Krox-24 [GCG(G/T)-GGGCG] for the formation of specific DNA-protein complexes. Our observations suggest that basal transcription of the A-chain gene in renal epithelial cells is achieved through active enhancement, mediated by a GC-rich PRE and nuclear proteins that bind to Sp-1-like consensus DNA sequences.

Animals↗

PubMed1994

Intracranial suppuration: a modern decade of postoperative subdural empyema and epidural abscess.

A retrospective study of subdural empyema and epidural abscess spanning 11 years and encompassing 41 patients was performed, demonstrating that the clinical characteristics of intracranial suppuration have changed over time. Sinusitis and otitis media, previously the predominant etiologies, were predisposing factors in only 29% of patients. A prior craniotomy had been performed in 66% of cases and was the most common risk factor for abscess development. The postoperative patients were subjected to detailed analysis. Patients who had undergone a prior craniotomy were notable for the following features: older age, lack of fever, evidence of wound infection, frequent false-negative computed tomographic scans, and a high percentage of Gram negative aerobic organisms or skin flora as pathogens. The population at highest risk for abscess development ranged from 50 to 60 years old, older than in previous series. Older age and an advanced degree of encephalopathy were indicative of a poor prognosis. Patients with subdural empyema had a worse prognosis as well. Hyponatremia was a frequent complicating factor. A much greater percentage of Gram-negative aerobic bacteria were isolated than in previous studies. Computed tomographic scans, half of which were performed with intravenous contrast material, were nondiagnostic in 30% of patients. The mortality rate was 18.5%, and delay in treatment correlated with increased risk of poor outcome. All patients were treated with a craniotomy. Repeated operations were required in three patients and were associated with the development of intraparenchymal abscess.

Adolescent↗

PubMed1993

Myf-4 does not mediate AChR receptor subunit mRNA expression in thymic tissues.

We investigated whether a MyoD gene family member plays a role in the expression of AChR or AChR-like proteins in human thymus. We amplified from thymic tissue, mRNA of subunits of the fetal- and adult-type AChR, and Myf-4, a gene product that appears to regulate AChR expression. RNA extracted from paraffin-embedded thymic tissue of five myasthenics and five nonmyasthenics was subjected to reverse transcription followed by polymerase chain reaction using primers specific for these mRNAs. Thymic RNA from eight of ten patients contained transcripts of the alpha- and epsilon-subunits (specific for the adult-type AChR). Presence of these transcripts did not correlate with thymic pathology or clinical presentation. No gamma-subunit (specific for the fetal-type AChR) or Myf-4 transcripts were found. Our results indicate that mRNA for the adult-type AChR is expressed in thymic tissue and is expressed by mechanisms not involving Myf-4. Thymic AChR subunits may be the primary epitope that initiates the immune response in MG against the adult-type AChR.

Adult↗

PubMed1993

A cationic region of the platelet-derived growth factor (PDGF) A-chain (Arg159-Lys160-Lys161) is required for receptor binding and mitogenic activity of the PDGF-AA homodimer.

Platelet-derived growth factor-AA and -BB homodimers and -AB heterodimers bind with high affinity to the platelet-derived growth factor (PDGF) alpha-receptor. Basic polypeptides such as polylysine and protamine sulfate compete with PDGF for receptor binding, suggesting a role for ligand positive charge in the binding interaction. A pentapeptide amino acid sequence with a cationic tripeptide core is perfectly conserved between the A- and B-chains (Val158-Arg159-Lys160-Lys161-Pro162) and was therefore considered as a possible alpha-receptor-binding domain. We have investigated the functional importance of positive charge within this region of the PDGF A-chain by using site-directed mutagenesis to convert the cationic core amino acids to the acidic sequence triglutamic acid. cDNAs encoding wild-type (PDGF-AAwt) and charge mutant (PDGF-AAcm) proteins were expressed following stable transfection of Chinese hamster ovary cells. Proper assembly and secretion of PDGF-AAcm was verified by metabolic labeling with [35S]cysteine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis analysis under nonreducing and reducing conditions. PDGF-AAcm was secreted as two major species of disulfide-linked A-chain homodimers identical in molecular mass to those observed for PDGF-AAwt (32 and 35 kDa). Secreted PDGF-AAwt and PDGF-AAcm proteins were purified to homogeneity and subjected to structural and functional analyses. Compared to purified PDGF-AAwt, PDGF-AAcm displayed a marked reduction in both binding affinity for PDGF alpha-receptors and mitogenic activity in Swiss 3T3 cells. Large reductions were also observed in the ability of semipurified PDGF-AAcm to stimulate calcium influx and the production of inositol phosphates. Measurement of circular dichroism spectra of highly purified PDGF-AAcm and PDGF-AAwt revealed no significant difference in secondary structure. Collectively, these results indicate that the cationic Arg159-Lys160-Lys161 region plays a critical role in the biological activity of PDGF-AA by direct participation in ligand binding to the PDGF alpha-receptor.

Amino Acid Sequence↗

PubMed1993

Acetylcholine receptor subunit gene expression in thymic tissue.

It is controversial whether acetylcholine receptors (AChR) or AChR-like proteins exist in human thymus. To evaluate this question we isolated RNA from paraffin-embedded thymic tissue of 5 myasthenics and 5 nonmyasthenics. RNA was subjected to reverse transcription followed by polymerase chain reaction (RT-PCR) using primers specific for beta-actin, subunits of the fetal- and adult-type AChR, and Myf-4, a gene product which regulates AChR expression in muscle. beta-Actin transcripts were identified in specimens from 8 of 10 patients. In these 8 patients alpha- and epsilon-subunit transcripts were identified. Presence of these transcripts did not correlate with thymic pathology or clinical presentation. No gamma-subunit (specific for the fetal-type AChR) or Myf-4 transcripts were found. Our results indicate that mRNA for subunits of the adult-type AChR are expressed in thymic tissue by mechanisms not involving Myf-4. AChR subunits in pathological thymus may provide a target for immune attack in MG. However, as AChR subunits were found in thymus tissue from myasthenics and nonmyasthenics, the presence of AChR subunits in thymus alone is not sufficient to produce myasthenia gravis.

Actins↗