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H J Allen

Publications and source records attributed to H J Allen.

At least 19 recordsLinked to original sources
PubMed2001

Structure-based design of caspase-1 inhibitor containing a diphenyl ether sulfonamide.

A series of compounds was designed and prepared as inhibitors of interleukin-1beta converting enzyme (ICE), also known as caspase-1. These inhibitors, which employ a diphenyl ether sulfonamide, were designed to improve potency by forming favorable interactions between the diphenyl ether rings and the prime side hydrophobic region. An X-ray crystal structure of a representative member of the diphenyl ether sulfonamide series bound to the active site of caspase-1 was obtained.

Caspase 1↗
PubMed2000

Microsatellite instability in ovarian and other pelvic carcinomas.

Twenty-six cases of ovarian carcinoma and six cases of other pelvic neoplasms were analyzed for microsatellite instability (MSI) using frozen specimens, fluorescence technology, and four selected markers (D2S123 on chromosome 2, D18S58 on chromosome 18, BAT26 on chromosome 2, and BAT40 on chromosome 1). This procedure also allowed the detection of loss of heterogeneity (LOH) at the four selected loci. One of the cases of ovarian carcinoma exhibited MSI and this was evident at three loci. Of 44 informative loci, 7 exhibited LOH representing 3 cases of ovarian carcinoma, 3 of 4 cases of primary peritoneal carcinoma, and one case of unknown primary. These data support other findings that MSI is not a frequent occurrence in ovarian cancer; however, LOH is a more frequent event and may be a target for the development of diagnostic/prognostic procedures for ovarian and primary peritoneal carcinoma.

DNA, Neoplasm↗
PubMed1999

Mutation analysis of BRCA1, TP53, and KRAS2 in ovarian and related pelvic tumors.

Cancer may be viewed as a genetic disease resulting from critical mutations that disrupt normal cell growth. To characterize the involvement of the BRCA1 and TP53 tumor suppressor genes and of the KRAS2 protooncogene in gynecologic cancer, mutation analysis of these genes was conducted in pelvic tumors of 85 patients that included 49 epithelial ovarian carcinoma cases. The 85 pelvic tumors contained 5 tumors with BRCA1 mutations, 33 with TP53 mutations, and 1 with a KRAS2 mutation. Each of the BRCA1 and KRAS2 mutations, and 25 of the TP53 mutations, were in ovarian carcinomas. Four of the BRCA1 mutations were germline and 1 was somatic. The 4 patients with germline BRCA1 mutations had an early age of disease onset (33-48 years) relative to the mean age of onset (58 years) of all 49 ovarian carcinoma patients, and 3 of these 4 patients had a family history of ovarian or breast cancer. None of the 4 tumors with germline BRCA1 mutations had a KRAS2 mutation or a TP53 mutation, despite a 51% frequency of TP53 mutations in the 49 ovarian carcinomas. Three of the 4 tumors with germline BRCA1 mutations retained a wild-type BRCA1 allele. The tumor with the somatic BRCA1 mutation contained a TP53 mutation and had no evidence for wild-type BRCA1 and TP53 alleles. These data suggest that both BRCA1 and TP53 were inactivated in 1 of 49 ovarian carcinomas. Moreover, mutational inactivation of both BRCA1 and TP53 did not occur in 4 tumors with a germline BRCA1 mutation. It has been proposed that tumorigenesis in cells with a heterozygous BRCA1 mutation requires inactivation of the wild-type BRCA1 and TP53 alleles, which results in genomic instability and acquisition of mutations in protooncogenes. Clearly, mutational inactivation of TP53 and the wild-type BRCA1 allele in ovarian tumors with a heterozygous, germline BRCA1 mutation is not an absolute requirement for tumor formation. It is possible that these alleles may be inactivated by nonmutational mechanisms or that other tumor formation pathways exist.

Adult↗
PubMed1998

Correlation of TP53 mutations and p53 expression in ovarian tumors.

To characterize the involvement of the TP53 tumor suppressor gene in ovarian cancer, mutation analysis of exons 2-11 of TP53 and immunodetection of its protein product, p53, were done in 48 ovarian tumors. Normally, p53 is not immunodetectable. Missense TP53 mutations have been reported to result in p53 accumulation and detection, but mutations generating premature stop codons have not. Mutations were identified in 19 of 41 malignant tumors but not in 5 benign tumors and 2 tumors of low malignant potential. Fifteen of the 19 tumors with mutations also stained positively by immunohistochemistry or Western blot or both. They included 11 missense mutations, 1 in-frame duplication (474ins6), and 3 frameshift mutations generating premature stop codons. The three tumors with frameshifts also had a wild-type TP53 allele and displayed normal size but not truncated p53 by Western blot. This indicates that these tumors express wild-type p53. The significance of TP53 mutations in the development of the three tumors is questionable unless there is a mechanism for inactivating wild-type p53. Nine of the 19 mutations found here, including the 3 frameshifts, were previously not reported in ovarian cancer. Thirteen of the 19 mutations were single nucleotide substitutions with 6 transitions and 7 transversions. The ratio of transversions to transitions (1.2) was different from literature reports (0.5) (P < 0.01). Thus, the spectrum of TP53 mutations in our study differed from other ovarian tumor reports. This difference may be due to population-based differences in the molecular epidemiology of TP53 mutations.

Blotting, Western↗
PubMed1998

Mutations in BRCA1 from fixed, paraffin-embedded tissue can be artifacts of preservation.

DNA isolated from paraffin-embedded tissues has been used for analysis of DNA alterations in disease states. Use of archival tissue can expedite the gathering of large numbers of specimens from rare disease subtypes that would take years to accumulate prospectively. Therefore, archival tissues from 70 ovarian cancer cases diagnosed before or at age 40 were retrieved for analysis of BRCA1 mutations. DNA was isolated from paraffin-embedded tissue of 70 ovarian cancer cases diagnosed before or at age 40. BRCA1 mutation analysis was conducted by single-strand conformation polymorphism analysis and DNA sequencing. Fifty-eight BRCA1 mutations were found in 34 of the 70 ovarian cancer cases. Twenty-two cases had one mutation each and 12 cases had multiple mutations. Multiple mutations found in histologically normal tissue of 2 cases were not present in matched tumor tissue. For another case, DNA from two separate blocks of normal tissue contained different mutations. These observations were anomalous and suggested that mutations detected in fixed tissues may be artifacts of tissue preservation and not present in the original unfixed tissues. To test this suggestion, blood was obtained from 2 patients for whom mutations were found in fixed, normal tissue. DNA from their unfixed lymphocytes did not contain the mutations found in fixed normal tissue. Thus, mutations found in fixed, paraffin-embedded tissues can be artifacts of tissue preservation. The reliability of DNA sequence data derived from such tissues must be questioned in the absence of corroborating data from unfixed tissues. This severely limits the use of fixed tissues as a source of DNA for retrospective research and for clinical genetic testing in families for which a disease-affected member is not alive.

Adult↗
PubMed1998

Binding of synthetic sulfated ligands by human splenic galectin 1, a beta-galactoside-binding lectin.

The carbohydrate-binding site of galectin 1, a vertebrate beta-galactoside-binding lectin, has a pronounced specificity for the betaGal(1-->3)- and betaGal(1-->4)GlcNAc sequences. The binding inhibition study reported herein was carried out to determine whether sulfation of saccharides would influence their binding by galectin 1. The presence of 6'-OSO3- on LacNAc greatly reduces the inhibitory potency relative to LacNAc. 3'-OSO3-LacNAc, 3'-OSO3-Galbeta(1-->3)GlcNAc(beta)1-OBzl and 3-OSO3-Galbeta1-OMe are more potent inhibitors than the non-sulfated parent compounds. Surprisingly, 2'-OSO3-LacNAc showed over 40 fold less inhibitory potency relative to LacNAc. Ovarian carcinoma A121 cells were shown to synthesize sulfated macromolecules that bind to galectin 1. Modulation in vivo of saccharide sulfation may lead to modulation of galectin 1 interaction with glycoconjugates; hence, sulfation could play a role in modulating lectin functions.

Asialoglycoproteins↗
PubMed1998

Genetic protocols review by Institutional Review Boards at National Cancer Institute-designated cancer centers.

Absent a clear and enforceable national policy on cancer genetic testing and genetic research, the responsibility may currently be on institutional review boards to regulate those activities at their institutions through protocol approval or disapproval. The survey reported here was carried out to gain information on National Cancer Institute-designated cancer center policies governing institutional review board review of genetic protocols and the preparedness of institutional review boards to review genetic protocols. Thirty-five responses (63% response rate) were received, of which 30 were evaluable. Twenty-four responders reported that they believed that there is a need for research that may lead to improved review of genetic research and genetic testing protocols. Only 14 responders felt adequately informed of current and developing issues and legislation relative to genetic testing and research. Seven responders reported that their cancer centers require an institutional review board-approved protocol for genetic testing activities. Five responders reported that their cancer centers have a formal written policy that guides institutional review board review of genetic testing protocols. About half of the responders reported that their cancer centers have no formal written policy that guides institutional review board review of genetic research protocols. Only three responders reported that institutional review board members receive formal training to prepare them to evaluate all of the issues associated with genetic protocols. We conclude that greater effort needs to be made to establish uniform policy governing cancer genetic testing and genetic research and greater effort should be made to prepare institutional review boards formally for the review of genetic-related protocols.

Cancer Care Facilities↗
PubMed1998

Assessment of family cancer history collection and utilization in patient care.

OBJECTIVES: This study was conducted to determine whether patients and accompanying persons visiting the Gynecologic Oncology Clinic were aware if a family cancer history was recorded and utilized in their medical care; whether they were aware of the importance of a family cancer history, and whether they would like to learn more about familial cancer. METHODS: Sixteen- and 17-item self-report questionnaires were administered to patients and their accompanying persons, respectively, who were visiting the Gynecologic Oncology Clinic. All responses were anonymous. RESULTS: Two hundred forty-four patient questionnaires and 114 accompanying person questionnaires were completed. Seventy-eight percent of the patients and 70% of the accompanying persons replied that a physician had inquired about their family history of cancer. Only 40% of those patients and 70% of those accompanying persons (31 and 49% of total patients and accompanying persons, respectively) replied that the inquiry was by their family physician. Sixty-seven percent of these patients and 63% of these accompanying persons reported that a written record was made of the family history. Thirty-one percent of the patients and 28% of accompanying persons knew that their family cancer history information had been used to aid in their medical care. Eighty-eight percent of the patients and 83% of the accompanying persons reported the occurrence of at least one relative with cancer; however, only 44% of the patients and 35% of the accompanying persons replied that a health care provider had ever provided teaching about the importance of a family cancer history. Seventy-five percent of the patients and 73% of the accompanying persons indicated that they would like to learn more about hereditary cancer and cancer genetics. CONCLUSIONS: This study demonstrates that patients desire information about cancer genetics and hereditary cancer. Therefore, health care providers should provide better education and information to their patients as well as improve their family history-taking skills.

Journal Article↗
PubMed1995

Isolation of a melibiose-binding protein from human spleen.

A melibiose-binding protein was isolated from human spleen by serial affinity chromatography on lactose-, mannose-, and melibiose-Sepharose. The purified protein agglutinated rabbit erythrocytes and re-bound to melibiose, but did not bind to murine nor human laminin. The protein was composed of approximately 58 kDa and 26 kDa polypeptides. The polypeptides were detected in buffy coat cell extracts and they were synthesized in vitro by B lymphoblastoid cells. The polypeptides did not react with anti-galaptin, anti-C-reactive protein, anti-amyloid P, anti-keratin, and anti-rat lung lectin 29 sera. The 58 kDa polypeptide reacted very weakly with anti-core-specific lectin serum and reacted with anti-IgG serum. The data suggest that the major protein isolated is an anti-Ga1 alpha 1-->6 immunoglobulin.

Animals↗
PubMed1993

Galaptin-mediated adhesion of human ovarian carcinoma A121 cells and detection of cellular galaptin-binding glycoproteins.

Previously, we have shown that galaptin, an endogenous beta-galactoside-binding lectin, is present in extracellular matrix where it may participate in the adhesion of A121 human ovarian carcinoma cells to extracellular matrix via interaction with specific cell surface carbohydrate receptors. We now report that A121 cells adhere to polystyrene plates coated with polymerized human splenic galaptin. The carbohydrate-mediated specificity of this adhesive interaction was demonstrated by inhibition with lactose. Additionally, treatment of A121 cells with neuraminidase increased cellular adherence by 30%, while beta-galactosidase treatment of cells decreased adherence by 65%. These findings prompted us to isolate and identify the cell surface galaptin receptor. In a Western blot of A121 cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 125I-labeled polymerized galaptin bound [corrected] to a unique cellular protein having a molecular mass of 110 kDa. This receptor was enriched by affinity chromatography using polymerized galaptin-Sepharose. Treatment of this material with N-glycanase ablated its galaptin-binding activity. In related studies, A121 cells metabolically labeled with [3H]glucosamine demonstrated a radiolabeled polymerized galaptin-binding protein with an identical molecular mass of 110 kDa. These studies confirmed the glycoprotein nature of this putative endogenous cellular galaptin receptor. Further studies with antibodies directed against two lysosomal associated membrane proteins, lamp-1 and lamp-2, demonstrated specific reactivity in Western blots with the 110-kDa glycoprotein. Additionally, 125I-polymerized galaptin recognized a 110-kDa protein in Western blots of material immunoprecipitated from A121 cell lysates by lamp-1 and lamp-2 antibodies. Finally, indirect immunofluorescence using antibodies directed against lamps detected cell surface antigenicity. Therefore, lamp-1 and/or lamp-2 appear to be the putative cell surface receptors involved in the adhesion of ovarian carcinoma cells to extracellular matrix mediated by galaptin.

Antigens, CD↗
PubMed1993

Galaptin and galaptin-binding glycoconjugates in serum and effusions of carcinoma patients.

The objectives of this study were (1) to quantify galaptin, an endogenous lectin, and galaptin-binding glycoconjugates present in normal serum, (2) to determine if these components were altered in the serum and effusions of carcinoma patients with advanced disease, and (3) to determine if ovarian carcinoma cells synthesize and release soluble galaptin inhibitors. Serum from healthy females (n = 10) had a mean galaptin content of 96 +/- 40 ng/ml. Galaptin levels in carcinoma patient serum (n = 29) were depressed (mean 21 +/- 23 ng/ml; p < 0.0001). Galaptin was not detected in 6 of 8 ovarian carcinoma patient sera. Effusions (n = 17) had a mean galaptin content of 358 +/- 326 ng/ml. Assays involving inhibition of binding of galaptin-peroxidase conjugates to asialofetuin were carried out to evaluate the levels of galaptin-binding glycoconjugates in serum and effusions. The mean inhibition titer of normal serum (n = 12) was 75 +/- 38. Patient serum (n = 28) had an elevated inhibitor content (mean titer = 304 +/- 155; p < 0.0001). Effusions (n = 17) also had a higher inhibitor content relative to normal serum (mean titer = 247 +/- 202; p = 0.0037). Ovarian carcinoma cells isolated from effusions and cultured in vitro were shown to synthesize and release into the medium galaptin-binding glycoconjugates of molecular mass 100-200 kD. An ovarian carcinoma cell line, A121, released galaptin-binding glycoconjugates of molecular mass > or = 200 kD into the medium. The data presented show that the levels of soluble galaptin and galaptin-binding glycoconjugates in the serum of advanced cancer patients are perturbed relative to normal female serum.

Breast Neoplasms↗
PubMed1992

Lymphoblastoid cell adhesion mediated by a dimeric and polymeric endogenous beta-galactoside-binding lectin (galaptin).

Glutaraldehyde-polymerized human splenic galaptin, a beta-galactoside-binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative to native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2-, 4- and 12-membered species after reductive alkylation. Both forms of galaptin bound, at 4 degrees C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5 kDa subunit molarity, were 5 x 10(7) binding sites/cell with affinity constant Ka = 2.2 x 10(5) M for dimeric galaptin and 17 x 10(7) binding sites/cell with Ka = 3.4 x 10(5) M-1 for polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic-adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic-adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 30-50% by lactose for plastic-adsorbed polymeric and dimeric galaptin, respectively. Native fetuin bound to the adsorbed dimeric galaptin in a lactose-insensitive manner. These data indicate that cell surface receptor-galaptin interaction is carbohydrate specific whereas polystyrene-adsorbed galaptin may demonstrate protein-protein interactions with soluble ligands.

Asialoglycoproteins↗
PubMed1992

Isolation of lactose-binding lectins from axolotl (Ambystoma mexicanum).

1. Lactose-inhibitable hemagglutination activity was identified in extracts of axolotl (Ambystoma mexicanum) larvae. 2. Two types of lectin were isolated from extracts by affinity chromatography on lactose-Sepharose. 3. A thiol-independent lectin of subunit mol. wt 15 kDa and a thiol-dependent lectin of subunit mol. wt 18 kDa were identified. 4. The 15 kDa and a 18 kDa polypeptides were weakly reactive with polyclonal anti-human galaptin serum.

Ambystoma↗
PubMed1992

Identification and synthesis of a novel 15 kDa beta-galactoside-binding lectin in human leukocytes.

Knowledge of the identity, synthesis and secretion of beta-galactoside-binding lectins by leukocytes is of importance because lactosaminoglycans present at the leukocyte cell surface may be physiologically significant lectin receptors that could mediate autocrine or paracrine functions and/or cell adhesion. This paper presents data that show that a previously identified 15.5-16.5 kDa lactose-binding protein synthesized in vitro by human peripheral leukocytes is actually comprised of three different polypeptides. One of these is related to a novel 15 kDa lectin isolated from human spleen and which is synthesized by B lymphoblastoid cells. Spleen contains at least six lactose-binding polypeptides for which the carbohydrate-binding activity is independent of the presence of divalent cations and mercaptoethanol. The splenic 15 kDa polypeptide does not appear to be immunologically related to previously characterized beta-galactoside-binding lectins. It is separable from galaptin, another galactoside-binding lectin (subunit mol. wt 14.5 kDa) by chromatography on DEAE-Sephacel. Western blot analyses and immunoprecipitation/fluorography experiments with metabolically labelled cells showed the presence of the 15 kDa lectin in peripheral leukocytes and in Epstein-Barr virus-immortalized B lymphoblastoid cells. The 15 kDa lectin yielded polypeptide fragments of approximately 6.2 and approximately 8.6 kDa after cyanogen bromide (CNBr) degradation. These fragments were partially sequenced and 12 residues/fragment were identified. A similarity search of the SWISS PROT protein data base did not reveal a relationship of the 15 kDa polypeptide to known lectins, including galaptin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Synthesis, isolation, and characterization of endogenous beta-galactoside-binding lectins in human leukocytes.

Galaptin, a beta-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5K), pI (4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonal antiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric beta-galactoside-binding protein of Mr 15.5-16.5K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chromatography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by the polymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived amino acids corresponded to residues 6-65, 84-114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Synthesis and carbohydrate-binding activity of poly(ethyleneglycol)-Ricinus communis agglutinin I conjugates.

The synthesis of poly(ethyleneglycol) (PEG)-lectin conjugates was investigated to provide new reagents for evaluation as biological response modifiers. PEG was activated with 1,1'-carbonyldiimidazole (CDI), followed by conjugation with Ricinus communis I (RCAI) lectin. The resulting conjugates were heterodisperse with respect to molecular weight. Carbohydrate-binding activity was retained. The conjugates were separated by affinity chromatography into fractions differing in apparent carbohydrate-binding affinity. Conjugation of RCAI with PEG 4 (mol.wt. 3350) or PEG 6 (mol.wt. 8000) appeared to provide less hindrance of the lectin binding site compared to conjugates prepared with PEG 20 (mol.wt. 20,000). Results of free amine assays indicated that higher ratios of PEG to RCAI in conjugates correlated with loss of low-affinity binding and retention of high-affinity binding. The data showed the feasibility of preparing PEG-lectin conjugates for in vivo use.

Binding Sites↗
PubMed1991

Localization of endogenous beta-galactoside-binding lectin as a means to distinguish malignant from benign skin tissue.

The immunolocalization of a 30-kd endogenous lectin, referred to as galaptin, was studied in various skin tumor types. Basal cell carcinomas expressed little or no galaptin, whereas nonmalignant basaloid cells, squamous cell carcinomas, melanoma, nevi, and stroma showed more prominent galaptin immunostaining. Therefore, the immunodetection of galaptin may aid the dermatopathologist in difficult histologic diagnosis.

Biomarkers, Tumor↗