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M J DiNubile

Publications and source records attributed to M J DiNubile.

At least 19 recordsLinked to original sources
PubMed2002

Changes in plasma gelsolin concentration during acute oxidant lung injury in mice.

Oxidant stress may contribute to acute lung injury under some circumstances. The rapid depletion of plasma gelsolin following major trauma in patients who subsequently develop respiratory distress suggests that this actin-scavenging protein might protect against delayed pulmonary complications. The specific aim of these experiments was to explore the temporal and quantitative relationship between gelsolin levels and lung damage. Gelsolin levels were measured in three murine models of oxidant injury: immunotargeting of pulmonary endothelium with an H2O2-generating enzyme; continuous exposure to >95% O2; and single high-dose thoracic radiation. The degree of lung injury was inversely related to gelsolin levels in mice treated with glucose oxidase-conjugated antibodies against platelet endothelial cell adhesion molecule-1 (p <0.0001). By 60-72 hours of hyperoxic exposure, gelsolin levels had dropped precipitously in all mice who sustained major lung damage (p <0.0001), establishing a quantitative association between gelsolin concentration and hyperoxic lung injury (r = -0.72; 95% confidence interval: ?0.81 to ?0.59). Gelsolin levels modestly but progressively fell in irradiated mice over the 3 days following treatment (p = 0.012) despite the development of only microscopic lung damage during this timeframe. These findings are consistent with the hypothesis that gelsolin depletion is involved in the pathogenesis of acute oxidant lung injury.

Animals↗
PubMed2002

Safety and tolerability of caspofungin acetate in the treatment of fungal infections.

Caspofungin acetate is the first member of the novel echinocandin class of antifungal drugs to be marketed in the United States. It has recently been approved for use in patients with invasive aspergillosis who are refractory to or intolerant of conventional therapy. Accordingly, its safety profile is particularly important to review. The safety and tolerability of caspofungin have been examined in 623 persons, including 295 patients who received >/= 50 mg/day for at least one week in clinical studies. In the 263 patients, given caspofungin in randomized double-blind active-control trials to date, there have been no serious clinical or laboratory drug-related adverse events; caspofungin was discontinued in only 2% of these patients because of drug-related adverse experiences. Caspofungin may have potentially important drug interactions with cyclosporine and tacrolimus.

Anti-Bacterial Agents↗
PubMed2001

A randomized double-blind study of caspofungin versus amphotericin for the treatment of candidal esophagitis.

Caspofungin is a new broad-spectrum antifungal drug. A multicenter, double-blind, randomized trial was conducted to assess the efficacy, safety, and tolerability of caspofungin relative to amphotericin B in adults with endoscopically documented symptomatic Candida esophagitis. By use of a modified intent-to-treat analysis, endoscopically verified clinical success was achieved in 74% (95% confidence interval [CI], 59%-86%) and 89% (95% CI, 72%-98%) of patients receiving caspofungin at 50 and 70 mg/day, respectively, and in 63% (95% CI, 49%-76%) of patients given amphotericin B at 0.5 mg/kg/day. Therapy was stopped because of drug-related adverse events in 24% of patients in the amphotericin B group and 4% and 7%, respectively, for the caspofungin groups. This report provides the first demonstration of clinical utility for an echinocandin compound. Caspofungin appeared in this study to be as effective as and better tolerated than amphotericin B for the treatment of esophageal candidiasis.

Adult↗
PubMed2001

Indinavir did not increase the short-term risk of adverse cardiovascular events relative to nucleoside reverse transcriptase inhibitor therapy in four phase III clinical trials.

A retrospective person-time analysis of the randomized and non-randomized extension phases of four phase III trials was performed to assess the incidence of adverse cardiovascular events in 2680 HIV-infected patients receiving indinavir or nucleoside reverse transcriptase inhibitor therapy, or both. The observed rate of cardiovascular events was not increased in patients receiving indinavir-based regimens compared with therapy without a protease inhibitor. Extrapolation of these findings is limited by the brief length of therapy and the small number of cases.

Anti-HIV Agents↗
PubMed2000

Skepticism: a lost clinical art.

Under the contemporary avalanche of new biomedical discoveries, most physicians find themselves in a losing battle to keep up to date. An underappreciated but important consequence of this struggle has been an abdication of the traditional responsibility of clinicians to critically review novel findings. We appear as a group to prefer our data predigested. Understandably, but unfortunately, we have become too accepting of formal guidelines and trusting of expert advice. Imprecision in our thinking and misapplication of cutting edge technologies have resulted, often to the detriment of the individual patient and the society at large. This essay illustrates some specific examples that demonstrate how superficial knowledge and blind faith can promote suboptimal care and inappropriate use of resources. Reflexive approaches to problems erode the individuality of diagnostic and treatment plans. Accordingly, I challenge my colleagues to be more skeptical about the validity and utility of how they are directed to practice medicine.

Clinical Competence↗
PubMed1999

Erythrocyte membrane fractions contain free barbed filament ends despite sufficient concentrations of retained capper(s) to prevent barbed end growth.

Many cellular functions depend on rapid cytoskeletal rearrangements localized to specific cytoplasmic domains. Tight regulation of the submembranous microfilament network is accomplished in large part in erythrocytes and granulocytes by actin binding proteins that cap the fast-growing barbed filament ends. Study of this dynamic system is necessarily hampered by the confounding perturbations of cell lysis and dilution. In this paper, we characterize the functional properties of the membrane-associated spectrin-actin complex from human erythrocytes as it exists after hypotonic lysis. Purified spectrin-actin "seeds" extracted from erythrocyte membranes effectively nucleated actin elongation from their barbed ends. However, polymerization from spectrin-actin complexes associated with the membrane fraction prematurely slowed despite the presence of G-actin in great excess of the critical monomer concentration. The addition of cytochalasin B decreased (rather than augmented) the slowing of elongation attributable to the membrane fraction, indicating that capping of barbed filament ends (not monomer sequestration) was the major mechanism underlying this effect. The paradoxical implication of our findings is that, despite the presence of excess capper(s) in the membrane fraction, the membrane-associated spectrin-actin seeds were not capped until after dilution into physiological ionic strength buffer containing monomeric actin. Furthermore, by comparing the degrees of contamination of the extracted and membrane-associated spectrin-actin preparations, it appeared that recognized capping proteins (including gelsolin and capping protein beta2) were not the predominant cappers found in the membrane pellet after hypotonic lysis. We hypothesize that the barbed ends of membrane-associated spectrin-actin complexes, while not excluding actin monomers, may be selectively inaccessible to certain cappers (perhaps simply as the result of steric hindrance). Growth from such complexes in vivo could be limited by the availability of polymerization-competent G-actin.

Actin Depolymerizing Factors↗
PubMed1998

Nucleation and elongation of actin filaments in the presence of high speed supernate from neutrophil lysates: modulating effects of Ca2+ and phosphatidylinositol-4,5-bisphosphate.

Cell motility depends on the rapid growth of cortical actin filaments whose barbed ends are capped in the resting cell. High speed supernates (HSS) of dilute neutrophil lysates contain actin monomers and/or oligomers that can be induced to polymerize by certain stimuli. We questioned whether some of the actin remaining in the supernate after high speed centrifugation exists as occult nucleation sites which can elongate when uncapped. Phosphatidylinositol-4,5-bisphosphate (PIP2) may play a critical role as an intracellular messenger in cytoskeletal rearrangement after stimulation by removing cappers from barbed filament ends. The experiments reported here examine the separate and interactive effects of PIP2 micelles and micromolar [Ca2+] on the rates of nucleation and elongation of pyrenyl-G-actin in the presence of HSS. HSS slowed the nucleation and elongation rates of gel-filtered pyrenyl-G-actin polymerized at submicromolar [Ca2+]. Under these conditions, PIP2 only slightly increased the number of nucleation sites, but delayed the slowing of the elongation rate in the presence of HSS. Nucleating activity in HSS could be induced by the addition of micromolar [Ca2+] and totally abolished by immunoprecipitation of gelsolin from HSS; incubation of HSS with PIP2 at micromolar [Ca2+] slightly decreased the number of calcium-induced nucleation sites in the supernate. Incubation of HSS with PIP2 before the addition of calcium led to a greater reduction in Ca2+-inducible nucleation sites. HSS possessed more nucleation sites after simultaneous exposure to PIP2 and Ca2+, followed by chelation of Ca2+ with EGTA, than HSS preincubated at micromolar [Ca2+] without PIP2. At submicromolar [Ca2+], PIP2 only generated a few barbed end nucleation sites in the HSS, but lessened the gradual slowing of elongation seen with HSS in the absence of PIP2, presumably by preventing capping by capping protein-beta2 in the supernate. Pointed end nucleating sites in HSS, attributable to gelsolin, could be created by adding micromolar [Ca2+]. The preincubation of HSS with PIP2 in the absence of micromolar [Ca2+] decreased the number of Ca2+-inducible nucleation sites in the HSS. Under conditions mimicking the sequential rise and fall of cytosolic [Ca2+] after stimulation, PIP2 accelerated actin polymerization despite the inhibitory action of HSS by maintaining Ca2+-activated nucleation sites. These observations suggest that a possible role for PIP2 in modulating cytoskeletal growth in vivo may be to regulate nucleation sites activated by sequential changes in cytosolic [Ca2+].

Actins↗
PubMed1998

Amphotericin B lipid complex for invasive fungal infections: analysis of safety and efficacy in 556 cases.

The safety and antifungal efficacy of amphotericin B lipid complex (ABLC) were evaluated in 556 cases of invasive fungal infection treated through an open-label, single-patient, emergency-use study of patients who were refractory to or intolerant of conventional antifungal therapy. All 556 treatment episodes were evaluable for safety. During the course of ABLC therapy, serum creatinine levels significantly decreased from baseline (P < .02). Among 162 patients with serum creatinine values > or = 2.5 mg/dL at the start of ABLC therapy (baseline), the mean serum creatinine value decreased significantly from the first week through the sixth week (P < or = .0003). Among the 291 mycologically confirmed cases evaluable for therapeutic response, there was a complete or partial response to ABLC in 167 (57%), including 42% (55) of 130 cases of aspergillosis, 67% (28) of 42 cases of disseminated candidiasis, 71% (17) of 24 cases of zygomycosis, and 82% (9) of 11 cases of fusariosis. Response rates varied according to the pattern of invasive fungal infection, underlying condition, and reason for enrollment (intolerance versus progressive infection). These findings support the use of ABLC in the treatment of invasive fungal infections in patients who are intolerant of or refractory to conventional antifungal therapy.

Adult↗
PubMed1997

High concentrations of phosphatidylinositol-4,5-bisphosphate may promote actin filament growth by three potential mechanisms: inhibiting capping by neutrophil lysates, severing actin filaments and removing capping protein-beta2 from barbed ends.

Cell locomotion requires rapid growth of cortical actin filaments whose barbed ends are capped in the resting cell. Phosphatidylinositol-4,5-bisphosphate (PIP2) may play a critical role as an intracellular messenger in cytoskeletal rearrangement after stimulation. We have examined the effects of PIP2 micelles on the Ca2+-independent actin filament capping activity in high speed supernatants of neutrophil lysates which we had previously demonstrated to be almost entirely due to capping protein-beta2, a homologue of cap Z. High concentrations of PIP2 totally prevented the capping of exogenous spectrin-F-actin seeds by dilute supernatants of neutrophil extracts. Capping could also be inhibited, albeit less effectively, by PIP and PI, but not by other phospholipids. When incubated with filaments in the absence of supernatant, PIP2 increased the number of growing ends. PIP2 also uncapped previously capped actin filaments, as demonstrated by incubating supernatant-capped and uncapped seeds with and without PIP2 and then comparing the initial elongation rates after addition of pyrenyl-G-actin. Incubation of capped seeds with high concentrations of PIP2 increased the number of free barbed ends to a level comparable to that of the uncapped seeds exposed to PIP2. PIP2 caused uncapping to occur too quickly to be explained simply by the off-rate of capping protein-beta2, implying that PIP2 interacted directly with capping protein on the filament ends. In fact, PIP2 transiently uncapped capped seeds in the presence of excess free capping protein. From our data, we estimate that millimolar concentrations of PIP2 (almost 100-fold higher than the amount predicted from the effective concentration in purified systems) would be required to inhibit all the capping protein-beta2 in the cytosol. This discrepancy probably results, in large part, from sequestration of PIP2 by other PIP2-binding proteins in the cytoplasm. If PIP2 mediates differential cytoskeletal growth after chemoattractant stimulation in vivo, very high concentrations may be required subjacent to the plasma membrane for regional severing and uncapping of actin filaments to occur quickly near the perturbed membrane.

Actin Depolymerizing Factors↗
PubMed1997

Capping of the barbed ends of actin filaments by a high-affinity profilin-actin complex.

Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, including sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting actin polymerization when barbed ends are free. Most previous studies have focused on profilin itself rather than its complex with actin. A high-affinity profilin-actin complex (here called profilactin) can be isolated from a poly-(L)-proline (PLP) column by sequential elution with 3 M and 7 M urea. Profilactin inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibition of elongation was observed with spectrin-F-actin than gelsolin-F-actin seeds, suggesting that the major effect of profilactin was due to capping the barbed ends of actin filaments. Its dissociation constant for binding to filament ends was 0.3 microM; the on- and off-rate constants were estimated to be 1.7 x 10(3) M-1 s-1 and 4.5 x 10(-4) s-1, respectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did not slow the elongation rate of pyrenyl-G-actin from filament seeds. Capping protein could not be detected by Western blotting in the profilactin preparation, but low concentrations of gelsolin did contaminate our preparation. However, prolonged incubation with either calcium or EGTA did not affect capping activity, implying that contaminating gelsolin-actin complexes were not primarily responsible for the observed capping activity. Reapplication of the profilactin preparation to PLP-coupled Sepharose removed both profilin and actin and concurrently eliminated its capping activity. Profilactin that was reapplied to uncoupled Sepharose retained its capping activity. Phosphatidylinositol-4,5-bisphosphate (PIP2) was the most potent phosphoinositol in reducing the capping activity of profilactin. Dissociation of the tight profilactin complex may serve as a unique mechanism by which profilin helps regulate actin filament growth.

Actins↗
PubMed1997

Prophylactic use of antibiotics and vaccines in patients with rheumatologic disorders.

In patients with serious underlying medical conditions, preventive interventions are a prudent, cost-effective, but underused strategy that could lessen morbidity and even mortality. In particular, immunization status should be assessed in all patients with rheumatologic disorders. Tuberculin reactivity is optimally documented before initiation of steroid therapy. It is often easier (and wiser) to prevent an infection than to treat it in a compromised host.

Antibiotic Prophylaxis↗
PubMed1997

Temporal association between serum gelsolin levels and clinical events in a patient with severe falciparum malaria.

Actin is present in high concentrations in most eukaryotic cells and can polymerize into filaments under physiological buffer conditions. As a result of tissue injury and cell lysis, large quantities of actin are released locally and may obstruct the downstream microvasculature, causing further damage to already injured organs. It has been postulated that this mechanism contributes to the development of the adult respiratory distress syndrome and to the diverse complications of falciparum malaria. Actin scavenging proteins--e.g., gelsolin--counteract the effects of extracellular actin, but the capacity of these plasma proteins can be overwhelmed by massive tissue injury. We examined the temporal relationship between serum levels of gelsolin (and tumor necrosis factor-alpha) and the clinical findings for a patient with severe falciparum malaria. The level of gelsolin decreased and then increased as the patient's status first worsened and then improved. We could not determine whether gelsolin served a biologically important function in this patient's recovery or was simply an epiphenomenon of disease activity. Gelsolin levels may be an early prognostic indicator in patients with a systemic inflammatory response syndrome. Moreover, the potential therapeutic role of recombinant human plasma gelsolin in patients with delayed organ dysfunction that commonly follows a self-limited initial insult merits investigation.

Adult↗
PubMed1996

Purification and properties of a Ca(2+)-independent barbed-end actin filament capping protein, CapZ, from human polymorphonuclear leukocytes.

In human polymorphonuclear leukocytes (PMN), changes in the actin architecture are critical for the shape changes required for chemotaxis and phagocytosis. Barbed-end capping proteins are likely to regulate actin assembly in PMN. The previously identified barbed-end blocking proteins in PMN, gelsolin and CapG, require Ca(2+) to initiate capping of actin filaments. Because chemoattractants can stimulate PMN actin assembly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cytoplasmic extracts. A Ca(2+) -insensitive actin polymerization inhibitory activity was partially purified from human PMN [Southwick & Stossel (1981) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PMN capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca(2+), as assessed by both pyrenylactin elongation and depolymerization assays. Similar to the activity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KC1 concentration from 0.1 M to 0.6 M. The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles, with half-maximal inhibition occurring at 5.5 micrograms mL(-1). PMN capZ did not nucleate actin assembly, sequester actin monomers, or sever actin filaments. Quantitative Western blot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affinity for barbed filament ends, capZ is likely to play an important role in the calcium-independent regulation of actin filament assembly associated with PMN chemotaxis.

Actin Cytoskeleton↗