Search PubMed⌕ Search

Biomedical subjects

R G Watts

Publications and source records attributed to R G Watts.

At least 37 records · Page 2Linked to original sources
PubMed1994

Actin polymerization and leukocyte function.

The coordinated remodeling of the filamentous actin-based microfilamentous cytoskeleton via regulated polymerization and depolymerization of globular and filamentous actin is required for polymorphonuclear leukocyte motile functions including locomotion, shape change, phagocytosis, and adhesion. Significant new observations on the structure and function of distinct filamentous actin pools in polymorphonuclear leukocytes, the mechanisms of chemotactic peptide-mediated actin polymerization, the role of filamentous actin in polymorphonuclear shape change in suspension and on a surface, the identification and characterization of rare patients with polymorphonuclear motile defects and actin dysfunctions, and regulation of actin reorganizations by actin regulatory proteins, the phosphorylation or dephosphorylation states of proteins, and the second messengers--the phosphoinositides--were reported in the past year. These observations form the basis for an improved understanding of the cellular and molecular role of actin assembly in polymorphonuclear function and are the subject of this review.

Actins↗
PubMed1993

Mechanisms for actin reorganization in chemotactic factor-activated polymorphonuclear leukocytes.

Cytoskeletal structure in polymorphonuclear leukocytes (PMNs) is thought to reflect a simple equilibrium between two actin pools (globular [G]- and filamentous [F] actin). Recent description of two distinct F-actin pools in PMNs (Triton-insoluble [stable] and Triton-soluble [labile] F-actin pools) (Watts and Howard, Cell Motil Cytoskeleton, 21:25, 1992) suggest a tripartite equilibrium between these F-actin pools and G-actin and multiple possible mechanisms for polymerization. To study the contribution of each actin pool to actin dynamics in PMNs, changes in actin content of the Triton-soluble and -insoluble F-actin pools and G-actin in chemotactic factor (CTF)-activated PMNs were measured by NBDphallacidin binding and by gel scans of Triton-lysed PMNs. From 0 to 30 seconds after CTF activation, PMNs rapidly increase total (Triton-soluble + Triton-insoluble) F-actin content (maximum = 1.7- +/- 0.10-fold basal at 30 seconds). Concurrent measures of the actin content of individual actin pools (Triton-soluble and -insoluble F-actin and G-actin) show that at all times (0 to 30 seconds) only the Triton-insoluble F-actin pool grows (maximum = 2.81- +/- 0.73-fold basal at 30 seconds), whereas both the Triton-soluble and G-actin pools simultaneously decrease (50% decrease at 30 seconds). Concurrent growth of one F-actin pool (Triton-insoluble) and loss of another F-actin pool (Triton-soluble) emphasize the functional uniqueness of the F-actin pools and can occur only if the Triton-soluble F-actin anneals or cross-links filament-to-filament with the Triton-insoluble fraction or if the Triton-insoluble F-actin pool first depolymerizes to monomer, which is then added to the Triton-insoluble pool. Because from 0 to 30 seconds after FMLP activation G-actin never increases, but, like the Triton-soluble F-actin progressively decreases, the results suggest that F-actin growth results from simultaneous new filament growth by monomer addition to the Triton-insoluble F-actin and cytoskeletal remodelling by Triton-soluble F-actin annealing or cross-linking to Triton-insoluble F-actin. These findings offer important new insights into the mechanism(s) of actin polymerization in CTF-activated human PMNs.

Actin Cytoskeleton↗
PubMed1992

Combination chemotherapy with ifosfamide and etoposide is effective in the treatment of central nervous system metastasis of childhood neuroblastoma.

Childhood neuroblastoma is a neural crest-derived tumor that presents most commonly during this period of life. In disseminated form, it is resistant to cure by chemotherapy. The tumor tends to recur in diverse locations after an initial clinical response. The parenchyma of the central nervous system (CNS) is a rare location for metastatic disease and typically represents terminal disease spread. Therefore, effective therapy for CNS metastasis of neuroblastoma has not been reported. The authors describe the case of a child who had a large parenchymal CNS metastasis at the time of initial recurrence of Stage IV neuroblastoma. Chemotherapy with ifosfamide and etoposide resulted in complete resolution of this lesion.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1992

Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.

Actins↗
PubMed1992

Isolation and characterization of transforming growth factor beta response variants from human prostatic tumor cell lines.

In this study we examined the relation between the response to transforming growth factor beta (TGF beta 1) in vitro and the growth in vivo of 1-LN-PC3-1A (1-LN) human prostatic carcinoma cells. 1-LN cells resistant to the growth-inhibitory effects of TGF beta 1 were isolated after exposure to 2 ng/ml TGF beta 1 in an anchorage-independent growth assay. Cloning of TGF beta 1-resistant and -sensitive populations produced 2 clones (R2-6 and 1-LN clone 4), which maintained relatively stable resistance or sensitivity, respectively, in the absence of TGF beta 1 for up to 12 passages. Colony formation by the R2-6 cells in the presence of TGF beta 1 was 2-10 times greater than that of 1-LN clone 4, depending upon the TGF beta 1 concentration. Injection of 1 x 10(5) R2-6 cells into athymic nude mice produced tumors with a significantly shorter latency interval as compared with 1-LN clone 4 tumors (P < 0.0001). Western immunoblotting showed that higher levels of latent TGF beta 1 protein were secreted into the culture medium by 1-LN clone 4 cells. Acidified conditioned media from both clones inhibited mink lung epithelial cell DNA synthesis. Neutralizing monoclonal antibody to TGF beta 1 but not TGF beta 2 abrogated this inhibitory effect. Comparison of the different sensitive and resistant clones showed that in vitro sensitivity to TGF beta 1 and in vivo tumor latency interval were not invariably correlated. Thus, the TGF beta 1 response phenotype in vitro was not always predictive of growth delay in vivo.

Animals↗
PubMed1992

Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils.

Polymerization of actin has been associated with development of polar shape in human neutrophils (PMN). To examine the relation of filamentous actin (F-actin) distribution to shape change in PMN, we developed a method using computerized video image analysis and fluorescence microscopy to quantify distribution of F-actin in single cells. PMN were labeled with fluorescent probe NBD-phallicidin to measure filamentous actin and Texas red to assess cell thickness. We show that Texas red fluorescence is a reasonable measure of cell thickness and that correction of the NBD-phallicidin image for cell thickness using the Texas red image permits assessment of focal F-actin content. Parameters were derived that quantify total F-actin content, movement of F-actin away from the center of the cell, asymmetry of F-actin distribution, and change from round to polar shape. The sequence of change in F-actin distribution and its relation to development of polar shape in PMN was determined using these parameters. After stimulation with chemotactic peptide at 25 degrees C, F-actin polymerized first at the rim of the PMN. This was followed by development of asymmetry of F-actin distribution and change to polar shape. The dominant pseudopod developed first in the region of lower F-actin concentration followed later by polymerization of actin in the end of the developed pseudopod. Asymmetric F-actin distribution was detected in round PMN before development of polar shape. Based upon these data, asymmetric distribution of F-actin is coincident with and probably precedes development of polar shape in PMN stimulated in suspension by chemotactic peptide.

Actin Cytoskeleton↗
PubMed1992

Cerebrovascular effects and tumor kinetics after a single intratumoral injection of human recombinant interleukin-2 alone or in combination with intravenous chemotherapy in a rat model of glioma.

It is well documented that drug delivery into experimental and human brain tumors is limited by the variably intact blood-brain barrier (BBB) at the growing edge. The aim of the present investigation was to examine the histopathological changes that occur after a single intralesional injection of human recombinant interleukin-2 (rIL-2) into a growing glioma and determine whether the injection improved delivery of cytotoxic drug into the neuropil surrounding the site of lymphokine injection. Because an intracerebral injection of rIL-2 causes a temporary breakdown in the BBB, we hoped to enhance drug penetration into peritumoral areas of brain with an intact BBB by using the novel biomodulating effect of rIL-2 on the cerebral endothelial cells. The results demonstrated that an intralesional injection of 7.2 x 10(4) National Units rIL-2 on Day 7 after tumor inoculation did not accentuate the already increased cerebrovascular permeability produced by the glioma nor did rIL-2 trigger additional or aggravate neurological deficits in glioma-bearing rats. Before the administration of chemotherapy in vivo, the RT-2 glioma cells were tested for in vitro sensitivity by colorimetric assay. At 24 hours after exposure to either methotrexate (MTX), vincristine (VIN), or doxorubicin (DOX), no significant inhibition of metabolic activity was observed. In contrast, a timed pulsed of any drug for 5 minutes caused significant dose-dependent inhibition of RT-2 glioma cells at 48 hours to 5 days after drug administration. Animal models receiving an intralesional injection of rIL-2 followed 3 days later by an intravenous dose of 30 mg/kg MTX, 0.23 mg/kg VIN, or 10 mg/kg DOX demonstrated that only MTX combined with intralesional rIL-2 significantly inhibited intracranial proliferation of RT-2 glioma cells. Use of intralesional rIL-2 and intravenous chemotherapy, however, did not significantly increase survival in this animal model of glioma. These results show that the combination of cytotoxic drugs with intralesional rIL-2 can be safely applied in the management of glioma and may form a rational basis for additional pharmacological investigations of a wider assortment of chemotherapies in combination with rIL-2 for intracranial malignancies.

Animals↗
PubMed1991

Severe and fatal anthracycline cardiotoxicity at cumulative doses below 400 mg/m2: evidence for enhanced toxicity with multiagent chemotherapy.

Three cases of severe, progressive, and in two cases, fetal cardiomyopathy secondary to anthracycline chemotherapy are reported. All of the patients were receiving multiagent chemotherapy for extremity sarcomas consisting of doxorubicin, high-dose methotrexate, bleomycin, cyclophosphamide, dactinomycin, and cisplatinum at the onset of their congestive heart failure. Cardiomyopathy developed in each patient at cumulative anthracycline doses less than 400 mg/m2. These cases suggest that enhanced cardiotoxicity may occur when anthracyclines are used in combination with other agents such as cyclophosphamide, bleomycin, cisplatinum, and methotrexate and that cumulative anthracycline doses considered to be "safe" may need to be lowered in these circumstances.

Adult↗
PubMed1991

A quantitative study of the role of F-actin in producing neutrophil shape.

Neutrophils change shape from round to polar and sequentially polymerize/depolymerize actin following chemotactic peptide activation in suspension. To study the relationship between changes in F-actin content and shape we altered the kinetics/extent of actin polymerization and depolymerization with tBOC peptide, cytochalasin D (CD), and low-dose FMLP, and determined the effect of these alterations on the temporal sequence of changes in neutrophil shape. F-actin was measured by FACS analysis of NBDphallacidin-stained cells and expressed as relative fluorescent intensity (RFI) compared to control (RFI = 1.00). Shape was determined by scanning electron microscopy. FMLP causes serial polymerization/depolymerization of actin (RFI = 1.00 +/- 0.04, 1.60 +/- 0.21, 1.10 +/- 0.18, and 1.05 +/- 0.14) associated with four distinct shapes (round-smooth, round-ruffled, blebbed, and polar) noted at 0, 30, 90, 300 sec respectively. Since blebbed and polar shapes appear concurrent with depolymerization and following polymerization, we determined whether depolymerization is required for polarization of cells. The kinetics of depolymerization were: (1) accelerated by tBOC addition at 45 sec, and (2) slowed by high concentrations of FMLP (greater than 10-7M) (300 sec RFI = 1.46). Neither change altered the time course of shape change. To determine whether duration of actin polymerization defines shape, polymerization was halted by addition of tBOC at 5, 10, 20, 30 sec after FMLP to block actin polymerization and shape was monitored at 300 sec. TBOC added 5-20 sec after FMLP limited neutrophil shape change to the blebbed form, while tBOC addition 30 sec following FMLP resulted in a polar shape at 300 sec. To determine whether the extent of actin polymerization affects the shape change sequence, polymerization was limited by (1) inhibition of polymerization with CD, (2) exposure of cells to low concentrations of FMLP (less than 10-9 M), and (3) interruption of polymerization with tBOC. Actin polymerization to RFI less than 1.35-fold basal results in blebbed shape; polymerization greater than 1.35-fold basal yields polar shape. The data show: (1) the human neutrophil demonstrates intermediate shapes when activated by chemotactic peptide, (2) depolymerization of F-actin does not determine shape, and (3) blebbed shape appears when actin polymerizes for greater than 5 sec; polar shape with polymerization greater than or equal to 30 sec to RFI greater than 1.35-fold basal. The data suggest actin polymerization is required for, and extent of polymerization determines, the shape of human neutrophils.

Actins↗
PubMed1990

Fatal varicella infection in a child associated with thymoma and immunodeficiency (Good's syndrome).

An 8-year-old male underwent excision of an encapsulated thymoma. Four months later he presented with disseminated, fatal varicella. Evaluation of his immune status during the terminal illness revealed hypogammaglobulinemia and lymphopenia consistent with a diagnosis of Good's syndrome (immunodeficiency with thymoma). This is the first case of Good's syndrome reported in a child and the first case of fatal varicella associated with Good's syndrome. The combination of this rare pediatric tumor and immunodeficiency is discussed. Despite specific antiviral therapy, varicella remains a deadly disease in the immunocompromised host.

Chickenpox↗
PubMed1990

Accidental poisoning with a superwarfarin compound (brodifacoum) in a child.

The "superwarfarin" compounds are 4-hydroxy derivatives of coumarin that have increased activity and a longer duration of action than the parent compound. The superwarfarins are used widely in the United States as rodenticides and are effective against warfarin-resistant strains of rats. A chronic accidental ingestion of one of these products, brodifacoum, by a 7-year-old child who had bleeding and laboratory evidence suggestive of a vitamin K-related coagulopathy is reported. The bleeding manifestations were severe and prolonged, requiring 13 months for normalization of coagulation times. With a negative history of ingestion and despite clinical suspicion, documentation of superwarfarin poisoning was hampered by the lack of readily available assays for these compounds, even from the manufacturers. Brodifacoum was also identified in rat feces from the family home. This finding raises the concern of poisoning not only from ingestion of brodifacoum particles themselves, but also from a fecal-oral route. A review of the literature is presented and the implications of this case for the practicing physician are discussed.

4-Hydroxycoumarins↗
PubMed1989

Histopathological and blood-brain barrier changes in rats induced by an intracerebral injection of human recombinant interleukin 2.

Adoptive immunotherapy utilizing human recombinant interleukin 2 (rIL-2) in conjunction with lymphokine-activated killer cells has shown some efficacy in the treatment of various types of cancers, particularly renal carcinoma and melanoma. Intravenous administration of rIL-2, with or without lymphokine-activated killer cells, produces a variety of serious side effects and approximately one-third of the patients experience a decrease in neurological status. Our previous investigations in animals have indicated that a single intravenous injection of rIL-2 can compromise the integrity of the blood-brain barrier (BBB). The present study examined the histopathological effect and BBB changes in rats which occur after a single intracerebral injection of rIL-2, its excipient, or saline into the parietal lobe. Animals were killed at various intervals up to 8 days (1 hour after intravenous injection of horseradish peroxidase), and the brain tissue was sectioned and processed for light microscopy. All animals showed increased cerebrovascular permeability for horseradish peroxidase due to traumatic BBB disruption at 4, 12, and 24 hours after injection. Extravasation of horseradish peroxidase persisted at 3 and 8 days only in animals injected with rIL-2. Injections of rIL-2 led to an increased leukocytic infiltration into the injection site, perivascular cuffing, and localized edema by 24 hours, which continued to increase over the 8-day study period. These results suggest that a single injection of human rIL-2 into the brain of rats induces an influx of leukocytes into the brain and may contribute to the cellular events that perpetuate a trauma-induced compromise in the integrity of the BBB.

Animals↗