Mechanism of action and limited cross-resistance of new lipopeptide MX-2401.

MX-2401 is a semisynthetic calcium-dependent lipopeptide antibiotic (analogue of amphomycin) in preclinical development for the treatment of serious Gram-positive infections. In vitro and in vivo, MX-2401 demonstrates broad-spectrum bactericidal activity against Gram-positive organisms, including antibiotic-resistant strains. The objective of this study was to investigate the mechanism of action of MX-2401 and compare it with that of the lipopeptide daptomycin. The results indicated that although both daptomycin and MX-2401 are in the structural class of Ca²âº-dependent lipopeptide antibiotics, the latter has a different mechanism of action. Specifically, MX-2401 inhibits peptidoglycan synthesis by binding to the substrate undecaprenylphosphate (C55-P), the universal carbohydrate carrier involved in several biosynthetic pathways. This interaction resulted in inhibition, in a dose-dependent manner, of the biosynthesis of the cell wall precursors lipids I and II and the wall teichoic acid precursor lipid III, while daptomycin had no significant effect on these processes. MX-2401 induced very slow membrane depolarization that was observed only at high concentrations. Unlike daptomycin, membrane depolarization by MX-2401 did not correlate with its bactericidal activity and did not affect general membrane permeability. In contrast to daptomycin, MX-2401 had no effect on lipid flip-flop, calcein release, or membrane fusion with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) liposomes. MX-2401 adopts a more defined structure than daptomycin, presumably to facilitate interaction with C55-P. Mutants resistant to MX-2401 demonstrated low cross-resistance to other antibiotics. Overall, these results provided strong evidence that the mode of action of MX-2401 is unique and different from that of any of the approved antibiotics, including daptomycin.

Consensus structure of Pf1 filamentous bacteriophage from X-ray fibre diffraction and solid-state NMR.

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.

Molecular structure of fd (f1, M13) filamentous bacteriophage refined with respect to X-ray fibre diffraction and solid-state NMR data supports specific models of phage assembly at the bacterial membrane.

Filamentous bacteriophage (Inovirus) is a simple and well-characterized model system. The phage particle, or virion, is about 60 angstroms in diameter and several thousand angstrom units long. The virions are assembled at the bacterial membrane as they extrude out of the host without killing it, an example of specific transport of nucleoprotein assemblages across membranes. The Ff group (fd, f1 and M13) has been especially widely studied. Models of virion assembly have been proposed based on a molecular model of the fd virion derived by X-ray fibre diffraction. A somewhat different model of the fd virion using solid-state NMR data has been proposed, not consistent with these models of assembly nor with the X-ray diffraction data. Here we show that reinterpreted NMR data are also consistent with the model derived from X-ray fibre diffraction studies, and discuss models of virion assembly.

Curling of flap tips in HIV-1 protease as a mechanism for substrate entry and tolerance of drug resistance.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) protease is an essential viral protein that is a major drug target in the fight against Acquired Immune Deficiency Syndrome (AIDS). Access to the active site of this homodimeric enzyme is gained when two large flaps, one from each monomer, open. The flap movements are therefore central to the function of the enzyme, yet determining how these flaps move at an atomic level has not been experimentally possible. RESULTS: In the present study, we observe the flaps of HIV-1 protease completely opening during a 10 ns solvated molecular dynamics simulation starting from the unliganded crystal structure. This movement is on the time scale observed by Nuclear Magnetic Resonance (NMR) relaxation data. The highly flexible tips of the flaps, with the sequence Gly-Gly-Ile-Gly-Gly, are seen curling back into the protein and thereby burying many hydrophobic residues. CONCLUSIONS: This curled-in conformational change has never been previously described. Previous models of this movement, with the flaps as rigid levers, are not consistent with the experimental data. The residues that participate in this hydrophobic cluster as a result of the conformational change are highly sensitive to mutation and often contribute to drug resistance when they do change. However, several of these residues are not part of the active site cavity, and their essential role in causing drug resistance could possibly be rationalized if this conformational change actually occurs. Trapping HIV-1 protease in this inactive conformation would provide a unique opportunity for future drug design.

Phase II study of vinorelbine in advanced and recurrent squamous cell carcinoma of the cervix.

PURPOSE: To evaluate the efficacy and toxicity of intravenous vinorelbine as single-agent chemotherapy in patients with advanced or recurrent squamous cell carcinoma of the cervix. PATIENTS AND METHODS: Between August 1993 and July 1995, 35 patients with advanced or recurrent squamous cell carcinoma of the cervix were entered onto this study. Patients had received no prior therapeutic chemotherapy. The initial dose of vinorelbine 30 mg/m2 was administered as a weekly intravenous infusion. Subsequent doses were unchanged, reduced, escalated, or omitted according to observed toxicity. Patients were evaluated for response and toxicity using standard Gynecologic Oncology Group (GOG) and World Health Organization criteria, respectively. RESULTS: Thirty-three of 35 patients were assessable for response and 35 of 35 for toxicity. The overall response rate was 18% (one complete response [CR], five partial responses [PR]). The mean response duration was 5.2 months and the median survival from treatment for all patients was 11.0 months. The major toxicity was leukopenia, with 61% of patients who had grade 3 or 4. Gastrointestinal and neurotoxicity were infrequent and mild. CONCLUSION: Vinorelbine has moderate activity in advanced or recurrent squamous cell carcinoma of the cervix. Further studies of combination regimens with this agent are justified in this patient population.

Intracerebral hemorrhage with rapid recovery.

Occasionally, patients with small supratentorial intracerebral hemorrhages exhibit the sudden onset of neurologic deficits, followed by rapid improvement during the next several days. We analyzed the computed tomographic (CT) and clinical features of a group of nine such patients, who were drawn from a series of 120 patients with intracerebral hemorrhage. The temporal pattern of illness was similar to that of a cerebral ischemic event, but the correct diagnosis of intracerebral hemorrhage was made on the basis of the CT findings.

Intracranial involvement of giant-cell arteritis.

Two cases of giant-cell arteritis with cerebral arteritis are presented. The clinical situation and the suggestive but nonspecific angiographic features make the antemortem diagnosis possible. Greater awareness of this entity will facilitate its diagnosis and the institution of effective steroid therapy.

Lumbar synovial cysts: report of eleven cases.

Over the past 18 months we have encountered 11 cases of symptomatic lumbar synovial cysts. This experience occurred during a period during which some 1,800 lumbar computed tomographic scans were done. The apparent increased incidence of these lesions is most likely due to the increased diagnostic ability made possible by the advent of high-resolution computed tomography and magnetic resonance imaging. This is a report and discussion of our 11 cases with a review of the literature. There is nothing distinctive in the physical findings or in the histories of our patients, but we have found, as have others, that high-resolution computed tomographic scanning and magnetic resonance imaging significantly enhance the diagnosis of such lesions.

Managing professional work: three models of control for health organizations.

Three arrangements for structuring the work of professional participants in professional organizations are described, contrasted and evaluated. Arguments are illustrated by application to the organization of physicians within hospitals. The primary rationale, the support structures that have fostered its development, the key structural features and the advantages and disadvantages of each arrangement are described. The effect on these arrangements of structures and forces external to any particular professional organization is emphasized.

An organizational field approach to resource environments in healthcare: comparing entries of hospitals and home health agencies in the San Francisco Bay region.

OBJECTIVE: To draw together insights from three perspectives (health economics, organizational ecology, and institutional theory) in order to clarify the factors that influence entries of providers into healthcare markets. A model centered on the concept of an organizational field is advanced as the level of analysis best suited to examining the assortment and interdependence of organizational populations and the institutional forces that shape this co-evolution. In particular, the model argues that: (1) different populations of healthcare providers partition fiscal, geographic, and demographic resource environments in order to ameliorate competition and introduce service complementarities; and (2) competitive barriers to entry within populations of providers vary systematically with regulatory regimens. DATA SOURCES: County-level entries of hospitals and home health agencies in the San Francisco Bay Area using data from the American Hospital Association (1945-1991) and California's Office of Statewide Health Planning and Development (1976-1991). Characteristics of the resource environment are derived from the Area Resource File (ARF) and selected government censuses. METHODS OF ANALYSIS: A comparative design is applied to contrast influences on hospital and home health agency entries during the post-World War II period. Empirical estimates are obtained using Poisson and negative binomial regression models. RESULTS: Hospital and HHA markets are partitioned primarily by the age and education of consumers and, to a lesser extent, by urbanization levels and public funding expenditures. Such resource partitioning allows independent HHAs to exist comfortably in concentrated hospital markets. For both hospitals and HHAs, the barriers to entry once generated by oligopolistic concentration have declined noticeably with the market-oriented reforms of the past 15 years. CONCLUSION: A field-level perspective demonstrates that characteristics of local resource environments interact with interdependencies of provider populations and broader regulatory regimes to affect significantly the types of provider organizations likely to enter a given healthcare market.

Effectiveness in professional organizations: the impact of surgeons and surgical staff organizations on the quality of care in hospitals.

In this research, we examine the relative importance of different structural units in a professional organization, the hospital, as they affect organizational effectiveness. The difficulties of measuring effectiveness in a complex professional organization are discussed, and an adjusted measure of surgical outcome is developed. Data are drawn from a prospective study of over 8,000 surgical patients treated by more than 500 surgeons in 15 hospitals throughout the nation. Two different types of analyses are presented, both indicating that hospital features have more impact on surgical outcomes than do surgeon characteristics. The second analysis assesses the relative importance of specific attributes of the hospital, surgical staff organization, and surgeon characteristics on surgical outcomes.

Hypertrophic synovitis and osteoarthritis of the cervical facet joint. Report of two cases.

We report two cases of cervical facet joint osteoarthritis associated with radiculopathy. Contrast-enhanced computed tomography showed a halo of enhancing soft tissue surrounding the facet joints and narrowing the neural foramen. Hypertrophic synovial masses were discovered and removed at the time of operation in each case, resulting in relief of the radicular symptoms.

Ocular injuries due to projectile impacts.

An animal model has been developed using enucleated porcine eyes to evaluate ocular trauma. The eyes were pressurized to approximately 18 mmHg and mounted in a container with a 10% gelatin mixture. The corneas of sixteen pressurized eyes were impacted by a blunt metal projectile (mass of 2.6 gm, 3.5 gm or 45.5 gm) at velocities of 4.0 to 38.1 m/s. The impacted eyes were evaluated by an ophthalmologist. A numerical classification scheme was used to categorize the severity of the ocular injury. A chi-squared test indicates that the injury level is associated with the kinetic energy (KE) and not the momentum of the projectile. The enucleated eyes began to experience lens dislocations when the KE of the projectile was approximately 0.75 Nm, and retinal injuries when the KE was approximately 1.20 Nm.