Biomedical subjects

D M Larson

Publications and source records attributed to D M Larson.

At least 19 recordsLinked to original sources

PubMed2006

Carbenoxolone inhibits junctional transfer and upregulates Connexin43 expression by a protein kinase A-dependent pathway.

We have been investigating the function and gene expression of connexins by vascular wall cells, especially Connexin43 (Cx43) in bovine aortic endothelial cells (BAEC). In this study, we tested the effects of carbenoxolone (CBN), a gap junction communication (GJC) blocker on the junctional transfer of Lucifer yellow in BAEC. CBN is a water-soluble derivative of the liquorice-root extract 18-alpha-glycyrrhetinic acid. CBN rapidly abolished dye-transfer in the scrape-load transfer assay (a measure of GJC) in a reversible and dose-dependent fashion. We then asked whether the BAEC might somehow compensate for the loss of junctional communication by altering the expression of connexins. Thus, we treated BAEC with 100 microM CBN in serum free medium and determined the total Cx43 cellular distribution (immunostaining) and protein content (immunoblotting). Besides changes in distribution, by 6 h, Cx43 content levels increased to 166% +/- 22% (P < 0.0001) of controls. RNA blot data showed two-three fold increases in Cx43 message in BAEC after 6 h of CBN treatment, suggesting transcriptional control. Since CBN has structural similarities to corticosteroids, we tested both aldosterone and prednisolone but neither drug increased Cx43 levels, suggesting that the CBN response was not due to a generalized steroid effect. Staurosporine inhibited the CBN-induced increase in Cx43 content, suggesting a role for kinases in the signaling pathway. Further studies with inhibitors indicated that PKA but not PKC was implicated. In summary, CBN blocks junctional communication and modulates Cx43 expression in BAEC. These results suggest a feedback mechanism for control of connexin expression based on junctional patency.

Aldosterone↗

PubMed2004

Glycosylphosphatidylinositol-specific phospholipase D immunoreactivity is present in islet amyloid in type 2 diabetes.

Numerous apolipoproteins associate with amyloid plaques. A minor high-density lipoprotein-associated protein, glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), has recently been described by the authors and others. Since GPI-PLD is synthesized by, and secreted from, pancreatic islet beta cells, the present study examined the hypothesis that GPI-PLD associates with islet amyloid. GPI-PLD immunoreactivity was examined in pancreatic tissues from type 2 diabetic and non-diabetic humans. GPI-PLD binding to heparan sulphate proteoglycan was determined in the absence or presence of heparan sulphate or heparin. Fibril formation from human islet amyloid polypeptide was determined in the absence or presence of GPI-PLD. In non-diabetics, GPI-PLD immunoreactivity was present and co-localized with insulin, as opposed to co-localizing with amyloid in diabetics. No immunoreactivity for apolipoprotein A-I was present in islet cells or islet amyloid. Heparan sulphate proteoglycan, which is commonly present in most amyloid, bound GPI-PLD in vitro. GPI-PLD inhibited the formation of amyloid fibrils from synthetic islet amyloid polypeptide in vitro. GPI-PLD is therefore present in islet amyloid and appears to derive from local production from islets. This localization likely derives from interaction between GPI-PLD and heparan sulphate proteoglycan. Since GPI-PLD also inhibited islet amyloid polypeptide fibril formation in vitro, it is concluded that GPI-PLD may play a role in islet amyloid formation in type 2 diabetes.

Amyloid↗

PubMed2001

Generation of RCAS vectors useful for functional genomic analyses.

Avian leukosis type A virus-derived retroviral vectors have been used to introduce genes into cells expressing the corresponding avian receptor tv-a. This includes the use of Replication-Competent Avian sarcoma-leukosis virus (ASLV) long terminal repeat (LTR) with Splice acceptor (RCAS) vectors in the analysis of avian development, human and murine cell cultures, murine cell lineage studies and cancer biology. Previously, cloning of genes into this virus was difficult due to the large size of the vector and sparse cloning sites. To overcome some of the disadvantages of traditional cloning using the RCASBP-Y vector, we have modified the RCASBP-Y to incorporate "Gateway" site-specific recombination cloning of genes into the construct, either with or without HA epitope tags. We have found the repetitive "att" sequences, which are the targets for site-specific recombination, do not impair the production of infectious viral particles or the expression of the gene of interest. This is the first instance of site-specific recombination being used to generate retroviral gene constructs. These viral constructs will allow for the efficient transfer and expression of cDNAs needed for functional genomic analyses.

Amino Acid Sequence↗

PubMed2001

TGF-beta1 induces an accumulation of connexin43 in a lysosomal compartment in endothelial cells.

We have been studying the relationships between cell growth and the expression of the gap junction protein Connexin43 (Cx43) in cultured bovine aortic endothelial cells (BAEC). As part of these studies, we examined the effect of the growth inhibitory cytokine TGF-beta1 on Cx43 expression. We have shown recently that TGF-beta treatment increases Cx43 mRNA and synthesis, content, and half-life of the protein within 24 h, which leads, over the course of days, to an accumulation of Cx43 in large, intensely immunostaining vesicles, filling much of the perinuclear cytoplasmic space. In the current study, based on their distribution and markers, we identified these vesicles as lysosomes/autophagosomes. Cx43 immunostaining and staining with a fluorescent probe for acidic compartments are coincident, as retention of a fluorescent-labeled low-density lipoprotein occurs in a similar pattern and the same staining pattern can be detected in the treated cells using other markers for lysosomal compartments. TEM revealed prominent lysosomal figures with considerable heterogeneous material. After withdrawal of TGF-beta, the accumulated Cx43 was cleared only slowly, with some brightly immunoreactive cells remaining even after 72 h. The prolonged appearance (based on immunoreactivity in situ and in immunoblots) of intact vesicular Cx43 in the treated cells suggests decreased degradation, resulting from impaired lysosomal activity. These data not only emphasize the importance of the lysosome in connexin degradation, but also show that TGF-beta can cause an alteration in lysosomal functioning, with implications for cellular metabolism.

Animals↗

PubMed2000

Functional expression and biochemical characterization of an epitope-tagged connexin37.

To study the gap junction protein connexin37 (Cx37), we stably transfected cell lines with constructs of human Cx37 containing the epitope tag FLAG (DYKDDDDK). A Cx37 construct containing the FLAG moiety at the carboxyl terminus (Cx37F) was expressed in BWEM cells, and did not substantially alter the levels of endogenous Cx43 in these cells. Immunostaining showed that Cx37F colocalized with Cx43 at cell-cell contacts. Pulse-chase metabolic labeling and immunoprecipitation with anti-FLAG antibodies indicated that Cx37F was synthesized as a protein that ran at 35.9 +/- 0.9 kDa on reducing SDS-PAGE but chased into a slower migrating band at 38.0 +/- 1.0 kDa. This shift in mobility was due to phosphorylation on serine residues, based on [(32)P]-metabolic labeling, immunoprecipitation, and phosphoamino acid analyses. The transition to the phosphoCx37F correlated with a loss of solubility in 1% Triton X-100. Based on the [(35)S]-methionine pulse-chase experiments, the half-life of the labeled Cx37F was approximately 3 h, which is within the range reported for other connexins. Analysis of dye injection experiments indicated that dye transfer was reduced in Cx37-transfected cells in comparison to parental BWEM cells, suggesting that formation of heteromeric Cx37-Cx43 channels reduced the molecular permeability of communication between these cells. Moreover, the similarities of previously demonstrated kinetic details and modification of Cx43 to our new data regarding Cx37 provide evidence for a commonality in processing and assembly steps of these two connexins.

Animals↗

PubMed2000

Benefit-Cost Analysis of StormwaterQuality Improvements.

The major purpose of this paper is to explore the potential value of benefit-cost evaluation for stormwater quality management decisions at a local level. A preliminary benefit-cost analysis (BCA) screening method is used for maximum extent practicable (MEP) analysis, identifying promising management practices, and identifying societal and economic tradeoffs for local stormwater problems. Ballona Creek, a major urban storm drain in Los Angeles, California, USA, is used to illustrate the practicality of the benefit-cost evaluation. The Ballona Creek example demonstrates the economic limits of stormwater management in an urban region and attests to the value of coordinated basinwide management compared to uncoordinated management by individual landowners. Evaluation results suggest that in urban areas, the benefit of stormwater quality improvements might be far greater if accompanied by comprehensive redesign of drainage networks and neighboring land uses. In this case, benefit-cost analysis is found to be useful for evaluating and understanding stormwater management alternatives despite the uncertainties in characterizing stormwater quality and the effects of stormwater management on improving receiving water quality.

Journal Article↗

PubMed2000

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins.

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ss1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

Animals↗

PubMed1999

Prognostic significance of DNA ploidy in endometrial cancer.

OBJECTIVE: The aim of this study was to analyze the prognostic significance of DNA ploidy in patients with endometrial cancer. METHODS: Between October 1988 and January 1997, DNA ploidy was determined prospectively in 208 women who were staged surgically by a standard protocol that included pelvic and para-aortic lymphadenectomy. Median follow-up was 48 months. RESULTS: Diploid tumors were identified in 154 (74%) patients and aneuploid tumors in 54 (26%). Patients with aneuploid tumors had a significantly higher prevalence of metastases to the cervix, adnexa, and omentum, malignant pelvic cytology, and advanced surgical stage. Patients with aneuploid tumors had a 4.5 times higher prevalence of pelvic lymph node metastases and a 5.8 times higher prevalence of para-aortic lymph node metastases. A significantly higher proportion of patients with aneuploid tumors was diagnosed with recurrent or progressive endometrial cancer (22.2 versus 6.5%, P = 0.002). Patients with aneuploid tumors had a significantly lower rate of survival from cancer death (P = 0.038) with 83% versus 94% surviving 5 years. CONCLUSION: Patients with aneuploid tumors are at high risk for lymph node metastases and should be surgically staged, including pelvic and para-aortic lymphadenectomy. Aneuploidy confers a risk for endometrial cancer death and these patients should be candidates for clinical trials evaluating treatment following surgery.

Aged↗

PubMed1998

Renal medullary carcinoma: report of a case with positive urinary cytology.

Renal medullary carcinoma is an aggressive neoplasm of the renal pelvis arising in patients with a history of sickle-cell trait. The authors report a case of renal medullary carcinoma with positive urinary cytology. Due to the location of the tumor in the renal pelvis and the loosely cohesive nature of poorly differentiated neoplasms, the presence of renal medullary carcinoma in a urinary cytology specimen is not surprising. The cytologic characteristics as well as the ultrastructural features are described.

Adolescent↗

PubMed1998

Surgical therapy for pulmonary aspergillosis in immunocompromised patients.

BACKGROUND: Medical management for invasive pulmonary aspergillosis (IPA) is often unsatisfactory. Antifungal therapy may be unable to eradicate IPA in the immunocompromised or neutropenic patient. METHODS: We retrospectively reviewed the surgical management of IPA in 13 immunocompromised patients at our institution. Twelve patients underwent perioperative bone marrow transplantation (4 autologous, 8 allogenic). All 13 patients received antifungal therapy. Eleven patients were neutropenic at the time of operation. RESULTS: The mean interval from diagnosis of aspergillosis to operation was 42 days (range, 3 to 135 days). Eighteen operations were performed on the 13 patients. Seven patients had resections from multiple pulmonary sites, whereas 6 had a single lesion resected. The average lesion resected was 3.7 cm in greatest diameter (range, 1 to 9 cm). After a mean follow-up of 21 months (range, 0 to 9 years), 3 patients (23%) are alive with no evidence of aspergillosis, 6 patients (46%) died without evidence of aspergillosis, and 4 patients (31%) died secondary to aspergillus infection. All 4 patients who died of aspergillus infection received an allogenic bone marrow transplantation. Two patients with direct extrapulmonic extension of IPA at time of operation died of recurrent aspergillus infections. Three of 4 patients who died of aspergillus infection had an absolute neutrophil count less than 1,300 cells/microL at time of operation. The mean absolute neutrophil count of the patients who cleared the aspergillus infection was 5,538 cells/microL. The mean survival of allogenic bone marrow transplant recipients was 5.2 months, and for recipients of autografts was 51.4 months. CONCLUSIONS: In this series, surgical resection of IPA cleared the aspergillus infection in 69% of the patients. Neutropenia, extrapulmonic extension of IPA, and allogenic bone marrow transplantation may predict a worse prognosis. Surgical resection of IPA in immunocompromised patients is an effective form of therapy in a properly selected patient population.

Adolescent↗

PubMed1998

Surgery without radiotherapy for primary treatment of endometrial cancer.

OBJECTIVE: To analyze the role of surgery alone, including pelvic and para-aortic lymphadenectomy, in patients with endometrial cancer who did not receive radiotherapy. METHODS: Between August 1987 and January 1997, 225 women with disease clinically confined to the uterus were staged surgically by a standard protocol that included pelvic and para-aortic lymphadenectomy in women with high risk factors. No radiation was administered before or after surgery. RESULTS: The combination of preoperative endometrial biopsy grade and gross depth of myometrial invasion identified 123 (55%) high-risk patients who had lymphadenectomy and 102 (45%) low-risk patients who did not. Eighteen (15%) high-risk patients had lymph node metastases and received postoperative systemic therapy. Three low-risk, eight high-risk-node-negative, and no high-risk-node-positive patients were diagnosed with recurrent cancer, corresponding to 5-year recurrence-free proportions of 0.95, 0.89, and 1.00, respectively. Although sample sizes and limited follow-up limit conclusions, the experience to date suggests a high rate of survival in all three groups. CONCLUSION: Our preliminary experience indicates that even high-risk patients have an excellent prognosis when treated with surgery, including pelvic and para-aortic lymphadenectomy, without radiotherapy.

Aged↗

PubMed1997

Differential regulation of connexin43 and connexin37 in endothelial cells by cell density, growth, and TGF-beta1.

We studied the growth-related expression of the gap junction proteins connexin43 (Cx43) and connexin37 (Cx37) to characterize mechanisms of their differential regulation in cultured bovine aortic endothelial cells. During growth to confluency, Cx43 mRNA levels were high in subconfluent cells and decreased at confluency; Cx37 mRNA was weakly detectable until the cultures became confluent, when Cx37 levels became similar to those of Cx43. Immunoprecipitation, immunoblots, and immunostaining demonstrated that Cx43 synthesis and content paralleled the changes in mRNA levels. These data suggested regulation of connexin expression related to growth status or cell density. We tested this hypothesis by inhibiting growth with transforming growth factor-beta1 (TGF-beta1). TGF-beta1 treatment caused an upregulation of Cx43 synthesis, content, and apparent half-life and an upregulation of mRNA, independent of changes in cell density. Increases in Cx43 synthesis preceded increases in mRNA, suggesting both translational and transcriptional regulation, whereas the increased half-life suggested post-translational regulation, as well. Immunostaining revealed the development of intense vesicular staining in the treated cells, which may explain the increased half-life. TGF-beta1 treatment also suppressed the upregulation of Cx37 expression. These alterations in connexin expression may have implications for endothelial communication under conditions of elevated vascular TGF-beta1 concentrations such as in wound healing.

Animals↗

PubMed1996

Prognostic significance of gross myometrial invasion with endometrial cancer.

OBJECTIVE: To determine if intraoperative estimation of gross myometrial invasion is sufficiently precise to guide surgical aggressiveness in staging patients with endometrial cancer. METHODS: Between September 1987 and September 1995, 236 women with endometrial cancer had visual estimation of gross myometrial invasion during surgical staging which included pelvic and para-aortic lymphadenectomy. RESULTS: In 213 patients (90.3%), the depth of gross myometrial invasion correctly predicted the microscopic depth of invasion on permanent histopathologic sections. Statistically significant associations were found between gross depth of myometrial invasion and tumor grade (P < .001), histopathology (P = .014), cervical metastases (P < .001), adnexal metastases (P < .001), omental metastases (P < .001), malignant pelvic cytology (P < .001), pelvic lymph node metastases (P < .001), para-aortic lymph node metastases (P = .001), and surgical stage (P < .001). Patients with more than 50% gross myometrial invasion were more likely to have poorly differentiated malignancies; nonendometrial histologies; malignant pelvic cytology; higher surgical stage; and cervical, adnexal, omental, pelvic lymph node, and para-aortic lymph node metastases. Patients with more than 50% gross myometrial invasion had a 6.4-fold higher prevalence of pelvic lymph node metastases, a 6.9-fold higher prevalence of para-aortic lymph node metastases, and a 6.7-fold higher prevalence of advanced surgical stage than patients with less than 50% myometrial invasion. CONCLUSION: Patients with endometrial cancer and more than 50% myometrial invasion on gross visual intraoperative estimation are at marked risk for extrauterine metastases, including pelvic and para-aortic lymph node metastases. Such patients should be considered for more aggressive surgical staging, including pelvic and para-aortic lymphadenectomy.

Adenocarcinoma↗

PubMed1996

Endogenous reactive oxygen metabolites mediate sublethal endothelial cell dysfunction during reoxygenation.

PURPOSE: Endothelial cells (EC) secrete vasoactive eicosanoids, which maintain organ blood flow. Because EC are a major source of eicosanoids, we studied the effects of reoxygenation on EC prostacyclin production. METHODS: Bovine aortic EC cultures were exposed to 2 hours of normoxia, then 1 hour of hypoxia (PO2 = 10 +/- 3.5 mm Hg), followed by 1.5 hours of reoxygenation in either normal medium or medium plus either superoxide dismutase (SOD, 300 units/ml), catalase (1200 units/ml), allopurinol (5.0 x 10(-4) mol/L), or dinitrophenol (10(-4) mol/L). RESULTS: Prostacyclin production decreased to 40% (p < 0.05) of basal prostacyclin production after 1 hour of hypoxia. EC reoxygenated with control medium recovered to 48% of basal prostacyclin production. EC reoxygenated in SOD resulted in recovery (p < 0.05) to 154% of basal prostacyclin production after 60 minutes. Catalase treatment resulted in recovery to 105% (p < 0.05) of basal prostacyclin production within 30 minutes of reoxygenation. Allopurinol treatment resulted in 77% recovery (p < 0.05) of basal prostacyclin production only during 30 minutes of reoxygenation. Dinitrophenol treatment resulted in significant (> or = 85%, p < 0.05) sustained recovery of basal prostacyclin production at 30, 60, and 90 minutes of experimental reperfusion. CONCLUSIONS: The hypoxia-induced decrease in EC prostacyclin does not recover during reoxygenation. Catalase/SOD allowed return to baseline prostacyclin production during reoxygenation, implicating reactive oxygen metabolites as mediators of decreased eicosanoid biosynthesis. Recovery of prostacyclin production after 60 minutes reoxygenation with dinitrophenol but not allopurinol suggests a mitochondrial origin of the oxygen metabolites responsible for decreased prostacyclin biosynthesis.

Analysis of Variance↗

PubMed1996

Glandular cells derived from direct sampling of the lower uterine segment in patients status post-cervical cone biopsy. A diagnostic dilemma.

Direct sampling of the lower uterine segment endometrium (LUS) may mimic atypical glandular lesions or resemble the cells of high grade squamous intraepithelial lesion. This study was done to further characterize the cytologic features of LUS and to determine the incidence of these cells in patients who have previously undergone cone biopsies. Cervical smears from 64 patients who had had a previous cervical cone biopsy were reviewed and compared to pre-cone cervical smears and any post-cone cervical biopsies or endocervical curettage specimens. Twelve smears from 63 patients (19%) were found to have fragments of LUS. Cytologic features of LUS included large groups with gland openings, branched glands, and nuclear palisading within the fragments. The fragments often were accompanied by endometrial stroma. Smaller fragments were frequently encountered. These were densely cellular but contained nuclear palisading.

Adolescent↗

PubMed1995

Comparison of D&C and office endometrial biopsy in predicting final histopathologic grade in endometrial cancer.

OBJECTIVE: To compare the accuracy of D&C and office Z-sampler endometrial biopsy in predicting hysterectomy tumor grade in women with endometrial cancer. METHODS: Between September 1987 and July 1994, 183 women with endometrial cancer had D&C or office Z-sampler endometrial biopsy before hysterectomy. RESULTS: One hundred thirty-one patients (72%) had Z-sampler biopsies and 52 (28%) had D&C. The Z-sampler correctly identified the hysterectomy tumor grade in 76 of 131 patients (58%), compared with 40 of 52 (77%) with D&C, a significant difference (P = .024). The major difference observed was an increased fraction of lesions undergraded (ie, a lower grade tumor found in the biopsy than in the hysterectomy specimen) by the Z-sampler (34 of 131, 26%) versus D&C (five of 52, 10%). CONCLUSION: Dilation and curettage was more accurate in identifying hysterectomy tumor grade and less likely to miss a higher-grade tumor than was Z-sampler biopsy. However, the inaccuracy of D&C alone necessitates further preoperative and intraoperative assessment for other risk factors to determine the aggressiveness with which an individual patient should be staged surgically.

Adult↗

PubMed1995

Immediate responses of endothelial cells to hypoxia and reoxygenation: an in vitro model of cellular dysfunction.

We have studied endothelial functions and integrity under clinically relevant levels of acute and profound hypoxia. Bovine aortic endothelial cells (EC) grown on microcarrier beads were exposed for 15-min intervals to normoxic (20% O2) or hypoxic (1-2% O2) medium. Control intervals were followed by four hypoxic and then four normoxic intervals for reoxygenation. Prostacyclin release from EC significantly decreased after only 15 min of hypoxia and remained low despite reoxygenation. This decrease in prostacyclin release was not coincident with decreased viable cells (Trypan blue exclusion) or with increased cell lysis (increased lactate dehydrogenase) after hypoxia or reoxygenation. When the medium was supplemented with 30 microM arachidonate (saturating concentration), prostacyclin release still significantly decreased after 30 min of hypoxia but returned to baseline levels by 30 min of reoxygenation. Similar results were obtained for thromboxane B2 release. These data suggest that 1) EC decrease prostacyclin release during acute, profound hypoxia, 2) EC decrease prostaglandin production during hypoxia despite abundant exogenous arachidonate, and 3) recovery of prostaglandin production is dependent on exogenous arachidonate during reoxygenation.

Animals↗

PubMed1995

High dose methotrexate infusion with leucovorin rescue for treatment of ectopic pregnancy.

The purpose of this article is to evaluate the effectiveness, side effects, and complications of high dose methotrexate infusion with leucovorin rescue in select patients with ectopic pregnancy. Between January 1991 and November 1994, 28 patients with ectopic pregnancies were prospectively treated with methotrexate (100 mg/m2 intravenous bolus followed by a 200 mg/m2 infusion over six hours) with leucovorin rescue. Twenty-seven of 28 patients (96%) were successfully treated. Only one patient (4%) required a second course of methotrexate to reach a normal hCG titer. One patient failed methotrexate infusion 45 days after treatment at a hCG titer of 12 mIU/mL. No Gynecologic Oncology Group grade 3 or 4 clinical, biochemical or hematologic toxicities occurred. Uterine bleeding and abdominal pain, not requiring transfusion or hospitalization, occurred in 71% and 56% of patients. The authors conclude that high dose methotrexate infusion with leucovorin rescue is a highly effective, well tolerated, nonsurgical treatment for select patients with ectopic pregnancy.

Abdominal Pain↗