Biomedical subjects

J R Howe

Publications and source records attributed to J R Howe.

At least 37 records · Page 2Linked to original sources

PubMed1998

The risk of gastrointestinal carcinoma in familial juvenile polyposis.

BACKGROUND: Familial juvenile polyposis (JP) is an autosomal dominant condition in which affected individuals develop upper or lower gastrointestinal (GI) juvenile polyps, or both, and have a predisposition to cancer of the gastrointestinal tract. The risk of GI cancer has not been well defined because of the small number of these families and the lack of follow-up. The objective of this study was to determine the prevalence and age at diagnosis of GI polyposis and cancer in a large JP kindred. METHODS: Medical records were reviewed, patients were interviewed, and histories were taken. Pathology reports and slides were reviewed by our pathologists. A database was created for analysis of clinical and pathologic factors. RESULTS: This kindred contains 117 members, 29 of whom have had upper or lower GI polyps or cancer, or both. All those affected have had colonic juvenile polyps or cancer, except for two who died of advanced gastric cancer and never had colonic evaluation. Nine individuals have had both upper and lower GI polyps or cancer. Sixteen of 29 (55%) affected patients have developed gastrointestinal cancer. Eleven (38%) have had colon cancer, and six (21%) have had upper GI cancers. CONCLUSIONS: The risk of gastrointestinal malignancy in affected members of this JP kindred exceeds 50%. The high risk of GI cancer warrants frequent endoscopic screening of both affected and at-risk family members. Screening will soon be facilitated by presymptomatic genetic testing for the identification of gene carriers.

Adenomatous Polyposis Coli↗

PubMed1998

Expression and heteromeric interactions of non-N-methyl-D-aspartate glutamate receptor subunits in the developing and adult cerebellum.

The localization and expression of ionotropic non-N-methyl-D-aspartate glutamate receptors (GluR) were investigated in the developing and adult rat cerebellum using subunit-specific polyclonal antibodies for immunocytochemical, immunoblot and immunoprecipitation studies. In P7 animals, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor immunoreactivity was detected in all layers of the cerebellar cortex with the exception of the external granule cell layer. Antibodies against the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits GluR1 and GluR4 gave strong immunoreactive staining of Bergmann glia in both young and adult animals, and both antibodies showed prominent staining of the molecular layer in the adult cerebellum. Dense immunoreactive staining of Purkinje cell somata and dendrites was obtained with anti-GluR2/3/4c in both the developing and adult cerebellum. Whereas each of the three alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antibodies stained the internal, but not the external, granule cell layer, immunostaining for the kainate-type subunits GluR6/7 and KA2 was detected in both the external and internal granule cell layers. as well as in the molecular layer in both P7 and adult cerebellum. Immunoblot analysis of total cerebellar protein indicated that the level of GluR4 expression increased 15-fold from P1 to P18, whereas the expression of the KA2 subunit protein was nine-fold lower in adult cerebellum than it was at P1. The expression of GluR1 increased moderately (two-fold) from P1 to adult. Subunit interactions between GluR1 and GluR4, as well as between GluR6/7 and KA2, were demonstrated in immunoprecipitation experiments; and the GluR4 and KA2 subunits appear to be present exclusively in heteromeric assemblies with GluR1 and GluR6/7, respectively. The results show that the various alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- and kainate-type subunits are differentially expressed during cerebellar development and further define the possible subunit composition of non-N-methyl-D-aspartate receptors in the major cerebellar cell types.

Animals↗

PubMed1998

A gene for familial juvenile polyposis maps to chromosome 18q21.1.

Familial juvenile polyposis (FJP) is a hamartomatouspolyposis syndrome in which affected family members develop upper and lower gastrointestinal juvenile polyps and are at increased risk for gastrointestinal cancer. A genetic locus for FJP has not yet been identified by linkage; therefore, the objective of this study was to perform a focused genome screen in a large family segregating FJP. No evidence for linkage was found with markers near MSH2, MLH1, MCC, APC, HMPS, CDKN2A, JP1, PTEN, KRAS2, TP53, or LKB1. Linkage to FJP was established with several markers from chromosome 18q21.1. The maximum LOD score was 5.00, with marker D18S1099 (recombination fraction of .001). Analysis of critical recombinants places the FJP gene in an 11.9-cM interval bounded by D18S1118 and D18S487, a region that also contains the tumor-suppressor genes DCC and DPC4. These data demonstrate localization of a gene for FJP to chromosome 18q21.1 by linkage, and they raise the possibility that either DCC or DPC4 could be responsible for FJP.

Adolescent↗

PubMed1998

Factors predictive of survival in ampullary carcinoma.

OBJECTIVE: To review the recent Memorial Sloan-Kettering Cancer Center experience with adenocarcinoma of the ampulla of Vater and to identify clinicopathologic factors that have an impact on patient survival. SUMMARY BACKGROUND DATA: The prognosis for patients with tumors of the ampulla of Vater is improved relative to other periampullary neoplasms. Identification of independent prognostic factors in ampullary tumors has been limited by small numbers of tumors and a lack of pathologic review. METHODS: Data were collected prospectively for patients presenting with periampullary carcinomas to the Memorial Sloan-Kettering Cancer Center between October 15, 1983 and June 30, 1995. The correlation between clinicopathologic variables and survival of ampullary carcinoma was tested by the Kaplan-Meier method and log-rank test, and Cox proportional hazards regression. Survival of patients with periampullary adenocarcinomas was compared by the Kaplan-Meier method. RESULTS: In 123 patients presenting with ampullary carcinoma, 101 tumors (82.1%) were resected. Factors significantly correlated with improved survival were resection (p < 0.01), and in resected tumors, negative nodes (p = 0.04) and margins (p = 0.02) independently predicted for improved survival. In periampullary tumors, the highest rates of resection and overall survival (median, 43.6 months) were found in ampullary carcinomas. CONCLUSIONS: Factors predictive of improved survival in ampullary carcinoma include resection, negative margins, and negative nodes. Improved overall survival in ampullary relative to periampullary adenocarcinoma is due in part to a significantly higher rate of resection.

Adenocarcinoma↗

PubMed1997

Characterization of RNA editing of the glutamate-receptor subunits GluR5 and GluR6 in granule cells during cerebellar development.

The non-NMDA class of ionotropic glutamate receptors are subject to RNA editing resulting in single amino acid changes within individual subunits that make up these oligomeric receptors. These amino acid changes result in significant alterations of important channel properties. Both edited and unedited versions of the kainate-receptor subunits GluR5 and GluR6 are present in brain, but whether this reflects the expression of both versions in individual types of neurons or differences in editing between different cell types is unclear. To characterize editing in a single identified type of central neuron, we have determined the extent to which GluR5 and GluR6 mRNAs are edited in acutely isolated cerebellar granule cells. RT-PCR analysis revealed that editing at each site in GluR5 and GluR6 increased during early postnatal development. The Q/R site was predominantly unedited in GluR5, whereas GluR6 was mostly edited. The Q/R and Y/C sites of GluR6 were edited to similar extents, whereas a smaller percentage of transcripts were edited at the I/V site. The expression of two double-stranded RNA adenosine deaminases implicated in GluR editing (DRADA and RED1) increased in granule cells between postnatal days 1 and 15. Finally, cerebellar granule cells express a previously unreported variant of RED1 which appears to arise from developmentally regulated alternative splicing.

Adenosine Deaminase↗

PubMed1997

The genetics of colorectal cancer.

This article reviews the genetic alterations that are thought to play a role in the development of sporadic and hereditary forms of colorectal cancer. It also highlights their potential utility in clinical practice, especially in the field of presymptomatic diagnostic testing for hereditary forms of colorectal cancer.

Adenomatous Polyposis Coli↗

PubMed1997

Introduction of the glutamate receptor subunit 1 into motor neurons in vitro and in vivo using a recombinant herpes simplex virus.

We developed and characterized a recombinant herpes simplex virus vector and used it to introduce the complementary DNA encoding glutamate receptor subunit 1 flip into postmitotic motor neurons. Infection of purified motor neurons in vitro with this vector resulted in selective, high-level expression of glutamate receptor subunit 1 immunoreactivity in nearly 100% of the neurons. Patch-clamp experiments demonstrated that the protein product of the glutamate receptor subunit 1 flip transgene assembles into functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels. Herpes simplex virus-glutamate receptor subunit 1 flip was introduced into spinal cord cells by direct injection into the ventral horn and selectively into motor neurons by sciatic nerve injection. High levels of expression were sustained for at least one week and were accompanied by changes in the ionic permeability of AMPA receptors in transgene-expressing neurons. Throughout the first week of infection, there was little evidence for toxicity. Herpes simplex virus provides a versatile tool for manipulating the glutamate receptor phenotype of postmitotic neurons and will permit study of the role of individual glutamate receptor subunits in neuronal physiology and pathophysiology.

Animals↗

PubMed1997

The molecular genetics of pancreatic cancer.

Pancreatic cancer is the fifth leading cause of cancer death in the United States, and despite improvements in the results of surgical treatment for this disease, little impact has been made upon overall mortality. New advances in treatment will depend upon improved adjuvant therapy, early diagnosis, and a better understanding of tumor biology. This article summarizes the results of molecular genetic studies in pancreatic cancer and their potential clinical significance. Familial predisposition to pancreatic cancer, cytogenic studies, DNA ploidy analysis, and examination of specific oncogenes and tumor suppressor genes are reviewed. The most frequent mutations detected have been in the K-ras oncogene, which occur in 80% of pancreatic cancers. These mutations do not correlate with tumor stage or survival, but can be useful in differentiating pancreatic exocrine from endocrine tumors and chronic pancreatitis. Mutations in the p53 gene occur in approximately 50% of tumors, and appear to be an independent prognostic factor for patient survival. Mutations in the CDKN2 gene are frequently seen in sporadic pancreatic cancers, and have been implicated in cases of familial pancreatic cancer. The significance of mutations in APC, MCC, DCC, c-erbB-2, RB-1, and mismatch repair genes in the genesis of pancreatic cancer is less clear.

Genes, p53↗

PubMed1997

DNA extraction from paraffin-embedded tissues using a salting-out procedure: a reliable method for PCR amplification of archival material.

Many techniques have been described for the extraction of DNA from paraffin-embedded tissues. Numerous efforts have been directed at simplification of these methods for rapid analysis using PCR. One disadvantage to some of the simpler procedures is inefficient PCR amplification, and for more involved ones using phenol/chloroform extraction, reduction in the yield of DNA. In the present study we report the use of a novel salting-out procedure that was utilized to extract DNA from 259 separate microdissection specimens of formalin-fixed, paraffin-embedded tissue sections. These sections were derived from 97 patients with tumors of the ampulla of Vater resected between 1965 and 1995 at our institution. The mean DNA yield was 22.75 micrograms (median 13.2 +/- 30.25) and the mean 260/280 absorbance ratio was 1.68 (median 1.70 +/- 0.25). All specimens (259/259) were successfully used to amplify K-ras exon 1 by a nested PCR technique. These results indicate that this DNA extraction method produces good yields of quality DNA, even from specimens several decades old.

Ampulla of Vater↗

PubMed1997

K-ras mutation in adenomas and carcinomas of the ampulla of vater.

The role of K-ras mutations in the progression of tumors of the ampulla of Vater is not well understood. To study the frequency and timing of K-ras mutations in ampullary tumors, areas of invasive carcinoma and adjacent adenomas were microdissected from paraffin blocks from 96 resected tumors. DNA was extracted, PCR amplification of K-ras exon 1 was performed, and PCR products were sequenced. Statistical analysis of K-ras mutations with respect to patient survival and clinicopathological factors was performed using the chi2 test, log-rank test, and Cox proportional hazard model. Thirty-four of 92 ampullary carcinomas (37.0%) and 25 of 46 adenomas (54.3%) had mutations in K-ras exon 1. Twenty-two of 23 (95.7%) adenomas adjacent to carcinomas with K-ras mutations also had K-ras mutations. The only clinicopathological factor significantly associated with K-ras mutation was tumor size >2 cm (P = = 0.035). Patient survival did not correlate with the K-ras mutation status (P = 0.31). We conclude that K-ras mutations are frequent in both adenomas and carcinomas of the ampulla of Vater and appear to occur as an early genetic event. The spectrum of mutations is similar to that observed in colorectal neoplasms, and these do not significantly correlate with patient survival.

Adenoma↗

PubMed1996

An evolutionarily conserved binding site for serine proteinase inhibitors in large conductance calcium-activated potassium channels.

Complementary DNA coding for the channel-forming alpha-subunit of a large conductance Ca(2+)-activated K+ channel (maxi Kca channel) was cloned from bovine aortic smooth muscle cells. This cloned mammalian KCa channel (Bslo) and its homolog from Drosophila (Dslo) were expressed in the HEK293 human embryonic kidney cell line. Both Bslo and Dslo KCa channels were sensitive to inhibition by the internally applied serine proteinase inhibitors: bovine pancreatic trypsin inhibitor (BPTI, KD = 7.0 microM for Bslo and 2.6 microM for Dslo) and chicken ovoinhibitor (OI, KD = 1.5 microM for Bslo and 11.4 microM for Dslo). BPTI and OI are members of the Kunitz and Kazal families of proteinase inhibitors, respectively. The approximately 60-residue inhibitory domains of these proteins have a different tertiary structure except in the region of a loop formed by approximately 6 residues, in which the peptide backbone adopts a similar conformation complementary to the active site cleft of many serine proteinases. At the single-channel level, BPTI and OI were found to inhibit KCa channels by a similar mechanism involving the production of discrete low-conductance events. These two inhibitors also exhibited competitive behavior, suggesting that they bind to an overlapping site. Kinetic characterization revealed that the dissociation rate of BPTI from the bovine KCa channel is fast (k(off) = 0.41 s-1), whereas that from the Drosophila KCa channel is slow (k(off) = 9.0 x 10(-4) s-1) and indicative of a strong molecular interaction. The stable complex of BPTI and trypsin was inactive as a KCa channel inhibitor, further supporting the idea that the trypsin inhibitory loop of BPTI recognizes a specific site on the channel protein. These results lead to the conclusion that the alpha-subunit of maxi KCa channels contains a conserved proteinase inhibitor binding site. We hypothesize that this site corresponds to a C-terminal domain of the channel protein that structurally resembles serine proteinases.

Amino Acid Sequence↗

PubMed1996

Cytotoxic effects of kainate ligands on HEK cell lines expressing recombinant kainate receptors.

Exposure of neurons either for prolonged periods of time or to high concentrations of excitatory amino acids (EAA), such as glutamate, results in neuronal death. Kainate also causes cell toxicity through the glutamate receptors. However, it is unclear whether the kainate receptor itself mediates any of the toxic responses. In the present study, HEK cells expressing the GluR6 +/- KA2 receptor subunit(s) were studied for their susceptibility to toxicity through the kainate receptor by kainate ligands. The natural ligand, glutamate, did not result in toxicity to the recombinant cell lines over that observed with the untransfected HEK cells, whereas kainate produced a 2-3-fold increase in LDH in both the HEK/GluR6 (ANOVA, P = 0.0001) and HEK/GluR6 + KA2 (ANOVA, P = 0.0002) cell lines following treatment with various dosages, but did not affect the HEK cells. Similar 2-3-fold increases in LDH activity were detected in both recombinant cell lines following treatment with 100 nM of SYM2081 ((2S,4R)-4-methylglutamic acid), a dose at which agonistic activity is elicited. The rank order potencies for eliciting toxicity are consistent with the previously reported EC50 values (SYM2081 > kainate > > > glutamate). Surprisingly, the kainate antagonist, NBQX, was the most toxic of the compounds tested although it had an affinity for the kainate receptor similar to glutamate. Treatment with as little as 10 nM elicited a dramatic increase in toxicity (6-10-fold) in the recombinant cell lines. At 1 microM, NBQX was significantly more toxic (Fisher PLSD, P < 0.05) than any of the other compounds tested. Thus, it appears that cell toxicity can be mediated via kainate receptor through two independent mechanisms: activation and blockage of the kainate receptor.

Cell Line↗

PubMed1996

Cloning of the cDNA encoding the sodium channel beta 1 subunit from rabbit.

Here, we report the nucleotide (nt) sequence of the cDNA encoding the sodium channel beta 1 subunit from rabbit (o beta 1). Cloning of the o beta 1 cDNA was accomplished by reverse transcription-polymerase chain reaction using rabbit brain RNA as a template for cDNA synthesis. The nt sequence of the o beta 1 cDNA predicts a 218-amino-acid polypeptide which is 96.3 and 97.3% identical to the beta 1 from human (h beta 1) and rat (r beta 1), respectively.

Amino Acid Sequence↗

PubMed1996

Homomeric and heteromeric ion channels formed from the kainate-type subunits GluR6 and KA2 have very small, but different, unitary conductances.

1. Patch-clamp methods were used to study recombinant glutamate receptor (GluR) ion channels expressed in human embryonic kidney cells (HEK 293) after transfection of the cells with cDNAs encoding kainate (KA)-type GluR subunits. Cells were transiently or stably transfected with the fully edited (R) version of GluR6 or they were contransfected with GluR6(R) and KA2. 2. Concentration-response data were obtained for KA and glutamate activation of homomeric GluR6(R) channels and fitted with Hill-type equations to give values for the agonist concentration at half-maximal activation (EC50), the Hill coefficient (nH), and the maximum current (Imax). Analysis of results obtained in seven cells with KA gave mean values of 0.47 microM and 1.47 for the EC50 and nH, respectively. The corresponding values for glutamate were 32 microM and 1.21 (n = 5). The Imax values obtained for KA and glutamate in the same cells were similar, suggesting that both KA and glutamate are full agonists at homomeric GluR6(R) channels. 3. Spectral density analysis of current noise evoked by KA and glutamate in GluR6(R)-expressing cells, or in outside-out patches from these cells, was used to obtain estimates of the apparent unitary conductance (gamma noise) of homomeric GluR6(R) channels. Results obtained with 10 microM KA in 15 cells gave a mean gamma noise value of 231 fS. The corresponding value from analysis of noise in outside-out patches was 259 fS (n = 4). Spectral density analysis of glutamate-evoked noise in six cells gave a mean gamma noise value of 264 fS. 4. Noise analysis of whole cell currents recorded in GluR6(R)/ KA2-expressing cell gave a mean gamma noise value of 572 fS (n = 9). Unlike homomeric GluR6(R) channels, GluR6(R)/KA2 heteromers were activated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) (0.25-5 mM). The mean EC50 for KA activation of GluR6(R)/KA2 channels was 1.62 microM (n = 6). 5. The results indicate that both homomeric GluR6(R) and heteromieric GluR6(R)/KA2 channels have a unitary conductance in the femtosiemen range. The coassembly of KA2 with GluR6(R) results in channels that have a different affinity for AMPA and KA and a two- to threefold larger unitary conductance.

Cell Line↗

PubMed1995

Cloning of a sodium channel alpha subunit from rabbit Schwann cells.

Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.

Alternative Splicing↗

PubMed1995

The responsiveness of a tetracycline-sensitive expression system differs in different cell lines.

A tetracycline-sensitive inducible expression system was used to regulate the expression of neurotransmitter receptor genes in two mammalian cell lines. The dopamine D3-receptor was stably expressed in GH3 cells, and GluR6 (a glutamate receptor subunit) was stably expressed in human embryonic kidney (HEK 293) cells. Three striking differences were found. 1) In the inactive state, virtually no D3-receptor expression was found in GH3 cells, whereas substantial levels of GluR6 expression were found in HEK 293 cells. 2) The induction of expression obtained upon removal of tetracycline was robust in GH3 cells but only modest in HEK 293 cells. 3) Whereas in each clonal cell line, the expression of a co-transfected hybrid transactivator is clearly regulated in a tetracycline-responsive manner, in the induced state, its mRNA levels were found to be very low in GH3 cells and very high in HEK 293 cells. The results indicate that, in contrast to GH3 cells, HEK 293 cells do not provide a cellular environment in which the expression of a heterologous gene can be tightly controlled in a tetracycline-responsive manner.

Animals↗

PubMed1995

Needle localization breast biopsy: a model for multidisciplinary quality assurance.

As part of the quality assurance role of the Cancer Committee at Barnes Hospital, an institutional audit of Needle Localization Breast Biopsy (NLBB) was performed. Mammographic, operative, and surgical pathology reports from 370 consecutive patients at our institution undergoing both mammography and needle localization biopsy over a 34-month interval were reviewed. Carcinoma was diagnosed pathologically in 103 patients (28%), and 27% of these proved to be noninvasive. Sixteen patients were found to have histologic or clinical involvement of the axillary nodes; no patients with Tis lesions were found to have axillary nodal involvement. Of the patients, 73% were found to have either Stage 0 or Stage I disease, and 61% with an established malignancy had mastectomy (67% of patients with invasive carcinoma, 44% of those with carcinoma-in-situ), whereas 39% had some form of conservation therapy (33% of patients with invasive lesions, 56% of those with carcinoma-in-situ). Our results have been compared with other published studies, and important clinical indicators for evaluating the results of individual centers performing NLBB are discussed. It is concluded that NLBB is a safe and effective method of biopsying nonpalpable breast lesions, which allows for the identification of early stage breast carcinomas. In the present environment of concerns about the quality of care and costs, it is the responsibility of each center performing NLBB periodically to evaluate their results with this multidisciplinary procedure and to bring about change in those areas found to be deficient.

Biopsy, Needle↗

PubMed1995

The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function.

Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotransfection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate studies of ion channel expression, localization, and processing.

Animals↗