Practical methods for the measurement of logP for surfactants.

Hydrophobicity is a commonly used parameter in quantitative structure activity relationships. The ability to determine the octanol-water partition coefficient (logP) empirically for non-ionizing, non-surfactant type chemicals using traditional stir-flask methods has been successful and well documented. In comparison the ability to measure logP for surfactants is considered impractical due to their amphiphilic nature, which gives them a tendency to form micelles and reside at the octanol-water interface. In this study we have shown that working with compounds below their critical micelle concentrations (CMC), at the experimental concentrations, it is possible to obtain experimental logP values for a series of sulphobetaines using the stir-flask method coupled with reversed phase-high performance liquid chromatography (RP-HPLC). Until now the ability to verify calculated logP values for surfactants has been limited. Measuring logP as described here can now be applied to other surfactants to validate existing and new modifications to the fragment method.

Hg-supported phospholipid monolayer as rapid screening device for low molecular weight narcotic compounds in water.

This study positions the fabricated Pt/Hg-supported phospholipid sensor element in the context of more conventional biomembrane-based screening platforms. The technology has been used together with immobilised artificial membrane (IAM) chromatography and COSMOmic simulation methods to screen the interaction of a series of low molecular weight narcotic organic compounds in water with phosphatidylcholine (PC) membranes. For these chemicals it is shown that toxicity to aquatic species is related to compound hydrophobicity which is associated with compound accumulation in the phospholipid membrane as modelled by IAM chromatography measurements and COSMOmic simulations. In contrast, the Hg-supported dioleoyl phosphatidylcholine (DOPC) sensor element records membrane damage/modification which is indirectly related to general toxicity and directly related to compound structure. Electrochemical limit of detection (LoD) values depend on molecular structure and range from 20 µmolL-1 for substituted phenols to 23 mmolL-1 for aliphatics. Rapid cyclic voltammetry (RCV) "fingerprints" showed that the major structural classes of compounds: alkyl/chlorobenzenes, substituted phenols, quaternary ammonium compounds and neutral amines interacted distinctively with the DOPC on Hg and that these observations correlated with and supported those predicted by the COSMOmic simulations of the compound/DMPC association. In addition, the compatibility of the electrochemical and COSMOmic methods validates the electrochemical device as a meaningful high throughput technology to screen compounds in water and report on the mechanistic details of their interaction with phospholipid layers.

Cholinergic mechanisms of cutaneous active vasodilation during heat stress in cystic fibrosis.

To test the hypothesis that cutaneous active vasodilation in heat stress is mediated by a redundant cholinergic cotransmitter system, we examined the effects of atropine on skin blood flow (SkBF) increases during heat stress in persons with (CF) and without cystic fibrosis (non-CF). Vasoactive intestinal peptide (VIP) has been implicated as a mediator of cutaneous vasodilation in heat stress. VIP-containing cutaneous neurons are sparse in CF, yet SkBF increases during heat stress are normal. In CF, augmented ACh release or muscarinic receptor sensitivity could compensate for decreased VIP; if so, active vasodilation would be attenuated by atropine in CF relative to non-CF. Atropine was administered into skin by iontophoresis in seven CF and seven matched non-CF subjects. SkBF was monitored by laser-Doppler flowmetry (LDF) at atropine treated and untreated sites. Blood pressure [mean arterial pressure (MAP)] was monitored (Finapres), and cutaneous vascular conductance was calculated (CVC = LDF/MAP). The protocol began with a normothermic period followed by a 3-min cold stress and 30-45 min of heat stress. Finally, LDF sites were warmed to 42 degrees C to effect maximal vasodilation. CVC was normalized to its site-specific maximum. During heat stress, CVC increased in both CF and non-CF (P < 0.01). CVC increases were attenuated by atropine in both groups (P < 0.01); however, the responses did not differ between groups (P = 0.99). We conclude that in CF there is not greater dependence on redundant cholinergic mechanisms for cutaneous active vasodilation than in non-CF.

Predicting the phospholipophilicity of monoprotic positively charged amines.

The sorption affinity of eighty-six charged amine structures to phospholipid monolayers (log KIAM) was determined using immobilized artificial membrane high-performance liquid chromatography (IAM-HPLC). The amine compounds covered the most prevalent types of polar groups, widely ranged in structural complexity, and included forty-seven pharmaceuticals, as well as several narcotics and pesticides. Amine type specific corrective increments were used to align log KIAM data with bilayer membrane sorption coefficients (KMW(IAM)). Using predicted sorption affinities of neutral amines, we evaluated the difference (scaling factor ΔMW) with the measured log KMW(IAM) for cationic amines. The ΔMW values were highly variable, ranging from -2.37 to +2.3 log units. For each amine type, polar amines showed lower ΔMW values than hydrocarbon based amines (CxHyN+). COSMOmic software was used to directly calculate the partitioning coefficient of ionic structures into a phospholipid bilayer (KDMPC-W,cation), including quaternary ammonium compounds. The resulting root mean square error (RMSE) between log KDMPC-W,cation and log KMW(IAM) was 0.83 for all eighty-six polar amines, and 0.47 for sixty-eight CxHyN+ amines. The polar amines were then split into five groups depending on polarity and structural complexity, and corrective increments for each group were defined to improve COSMOmic predictions. Excluding only the group with sixteen complex amine structures (≥4 polar groups, Mw > 400, including several macrolide antibiotics), the resulting RMSE for corrected KDMPC-W,cation values improved to 0.45 log units for the remaining set of 138 polar and CxHyN+ amines.

Species cross-reactive membrane-associated urothelial differentiation antigen.

An antigen, termed "urothelium membrane antigen" (UMA), confined to urothelium and most abundantly associated with the asymmetric unit membrane of the terminally differentiated luminal cells was identified with a monospecific (absorbed or affinity purified) rabbit antiserum obtained by immunization with membranes from normal bovine urothelium. A cross-species immunofluorescence-positive reaction was observed in normal bladder: Luminal urothelial membrane reacted most intensely. Underlying normal urothelial cells also showed a weaker cytoplasmic reaction. Specific luminal membrane labeling was confirmed by transmission and scanning electron microscopy. Specificity of the anti-UMA differed from the general epithelial reactivity obtained with antibodies to bovine urothelium cytosol. Anti-UMA did not cross-react with keratin, although there was some evidence of a filamentous localization in the cytoplasm of permeabilized cells. UMA was variably expressed by urothelial carcinoma-derived cell lines to a degree apparently related to the degree of cell differentiation, showing the highest positivity on the well-differentiated RT4 and RT112 lines and only a very weak reaction with the anaplastic MGHU-1 (EJ) and T24 lines. On immunoblots, anti-UMA reacted with a peptide of approximately 54,000 daltons.

Phospholipophilicity of CxHyN(+) amines: chromatographic descriptors and molecular simulations for understanding partitioning into membranes.

Using immobilized artificial membrane high-performance liquid chromatography (IAM-HPLC) the sorption affinity of 70 charged amine structures to phospholipids was determined. The amines contained only 1 charged moiety and no other polar groups, the rest of the molecule being aliphatic and/or aromatic hydrocarbon groups. We systematically evaluated the influence of the amine type (1°, 2°, 3° amines and quaternary ammonium), alkyl chain branching, phenyl ring positioning, charge positioning (terminal vs. central in the molecule) on the phospholipid-water partitioning coefficient (KPLIPW). These experimental results were compared with quantum-chemistry based three-dimensional (3D) molecular simulations of the partitioning of charged amines, including the most likely solute conformers, using a hydrated phospholipid bilayer in the COSMOmic module of COSMOtherm software. Both IAM-HPLC retention data and the simulations suggest that the molecular orientation of charged amines at the location in the bilayer with the lowest calculated Gibbs free energy exerts a strong influence over the partitioning within the membrane. The most favourable position of charged amines coincides with the region where the phosphate anions in the phospholipid bilayer are most abundant. Hydrocarbon units oriented in this layer are located more towards the aqueous phase and contribute less to the overall membrane affinity than hydrocarbon units extending into the more hydrophobic core of the bilayer. COSMOmic simulations explain most of the trends between the structural differences observed in IAM-HPLC based KPLIPW. For this set of cationic structures, the mean absolute difference between COSMOmic simulations and IAM-HPLC data, accounting only for amine type corrective increments, is 0.31 log units.

Characterization of the P-glycoprotein over-expressing drug resistance phenotype exhibited by Chinese hamster ovary cells following their in-vitro exposure to fractionated X-irradiation.

Exposure of the Chinese hamster ovarian AuxB1 cell line in vitro to fractionated X-irradiation generated sublines designated DXR-10, which proved resistant to multiple drugs and overexpressed P-glycoprotein (Pgp), as judged by Western blotting using the C219 monoclonal antibody. Further characterization of these irradiated DXR-10 sublines has provided evidence for: (i) the expression of cross-resistance to gramacidin D, taxol, puromycin and Navelbine, but not to daunomycin or mitoxantrone; (ii) overexpression of the class I Pgp, as judged by Western blotting using the C494 monoclonal antibody; (iii) decreased accumulation of 3H-vincristine, which could be enhanced by verapamil addition; (iv) unaltered accumulation and subcellular distribution of adriamycin; (v) significantly increased rhodamine 123 accumulation in the presence of verapamil; (vi) plasma-membrane ultrastructural modifications resulting in a significantly increased surface area; (vii) numerous clonal karyotypic alterations, with abnormalities involving the long arm of chromosome 1 being consistently identified; (viii) a lack of overexpression of sorcin; (ix) increased total glutathione levels and overexpression of glutathione S-transferase pi. The fact that only certain of these features are considered characteristic of the 'classic' multidrug-resistant CHRC5 cell line supports our earlier proposal that exposure to fractionated X-irradiation results in the expression of a unique drug-resistance phenotype.

Effect of exchange transfusion with Fluosol DA 20% on splenic distribution and immunocompetence of rat lymphocytes.

T- and B-splenic lymphocyte frequency, immune response 4 days after immunization with sheep red blood cells (SRBC), and proliferative response to concanavalin A (Con A) were determined 1, 3, and 5 days after exchange transfusion with Fluosol DA 20% (FDA) in adult, male Sprague-Dawley rats vs sham transfused rats. T-cell, T-helper, T-suppressor, and B-lymphocyte were reduced 1 day after transfusion (P less than or equal to 0.001). T- and B-lymphocyte frequencies were still reduced at day 3 (P = 0.0372). By day 5, there were no significant reductions in T-cell, T-helper, T-suppressor, and B-cell frequencies in the FDA-transfused rats. The frequency of cells with cytoplasmic IgG was reduced (P less than or equal to 0.025) in cells harvested from spleens of FDA-transfused rats and tested fresh. Proliferative response of splenic lymphocytes to Con A was unaffected by transfusion with FDA (P greater than or equal to 0.078). Splenic hemolytic plaques in response to SRBC were unaffected if rats were transfused 3 days after immunization with SRBC and 1 day prior to study (P = 0.941), enhanced if rats were transfused 1 day after SRBC immunization and 3 days prior to study (P = 0.0015), and suppressed if rats were transfused 1 day before SRBC immunization and 5 days before study (P less than 0.0001). Transfusion with FDA causes transient decreases in identifiable T and B lymphocytes, depresses cytoplasmic IgG-positive B cells, does not affect proliferative response to Con A, does not affect an ongoing specific immune response, enhances an early specific immune response, and inhibits the induction of a specific immune response.

Determination of serum gentamicin sulfate levels: ordering patterns and use as a guide to therapy.

Determination of serum gentamicin levels (SGLs) is helpful in monitoring therapy in patients receiving this drug because there is a narrow therapeutic margin and serum levels may vary greatly between patients receiving similar doses. During a ten-month period, definite indications for obtaining SGL determinations were present for 189 of 212 serum samples (89.2%) submitted to the laboratory. However, 110 samples (51.9%) were improperly drawn and results of 85 samples (40.1%) were ignored. Only 26 of the 62 correctly drawn and not ignored samples (41.9%) were appropriately acted on. At most, 42 of the 212 samples (19.8%) were appropriately used in making patient-care decisions. Thus, in this study, an inordinatley large number of SGLs were incorrectly drawn, apparently ignored, or inappropriately used in making patient-care decisions.

Gram-negative bacteremias. Analysis of factors for clinical assessment of gentamicin resistance.

We reviewed the charts of 163 patients with 183 episodes of Gram-negative bacillary bacteremia to determine a clinical profile that would select patients at high risk for experiencing gentamicin-sulfate-resistant Gram-negative bacillary bacteremia at our hospital. Gentamicin-resistant Gram-negative bacilli were only associated with institution-acquired bacteremia. Among institution-acquired episodes, urinary tract infection, diagnostic or therapeutic procedures of the lower respiratory tract or urinary tract, presence of pneumonic infiltrate on chest roentgenogram, prior therapy with gentamicin, and prior therapy with other antibiotics were significant risk factors. Because only two of the 29 gentamicin-resistant bacteria that were tested against amikacin base were resistant to amikacin, we advocate initial treatment with amikacin for patients with evidence of an institution-acquired Gram-negative bacteremic episode. Gentamicin is still our initial choice for a community-acquired episode.

Zygomycosis of the maxillary sinus and palate caused by Basidiobolus haptosporus.

Basidiobolus haptosporus is known to cause subcutaneous zygomycosis in tropical Africa and Asia. We cared for a 49-year-old hyperglycemic, asplenic man who had never traveled outside the United States and who was seen initially for a painless palatal ulcer with cutaneous hypesthesia of the right cheek and upper lip. An invasive process involved the right middle nasal turbinate, maxillary antrum, maxillary division of the trigeminal nerve, and bony palate. Histological examination of biopsy tissue showed necrotizing granulomata with broad, nonseptate hyphae. Basidiobolus haptosporus was cultured from this tissue. Various laboratory studies revealed no immunologic defect and his lesions responded to therapy with amphotericin B. To our knowledge, this represents the first case of zygomycosis caused by B haptosporus in the Americas and the first culture-documented case of invasive mycosis caused by this mold.

Mild nephrotoxicity associated with vancomycin use.

Nephrotoxicity related to vancomycin hydrochloride therapy has been reported at overall rates of 7% to 16% and as high as 35% when combined with an aminoglycoside antibiotic. We conducted a prospective study in older men. A group that received vancomycin was compared with a control group to determine the incidence of nephrotoxicity secondary to vancomycin therapy alone and in combination with aminoglycosides, to identify possible risk factors associated with nephrotoxicity, and to determine the incidence of other adverse effects associated with vancomycin use. Nephrotoxicity occurred in 11 (17%) of 66 patients receiving vancomycin and in 3 (5%) of 57 controls overall. Stepwise logistic-regression analysis failed to identify underlying illnesses or concurrent risks that may have contributed to the development of nephrotoxicity associated with vancomycin. Adverse effects, including phlebitis (14%), neutropenia (1%), rash (0%), and red neck syndrome (0%), occurred at rates similar to previous reports.

Pseudobacteremia caused by Clostridium sordellii.

Thirteen of 280 (4.6%) blood cultures collected over a 12-day period were positive for Clostridium sordellii, a spore-forming anaerobe, rarely considered a human pathogen. Nosocomial bacteremia and intrinsic contamination of material used to culture blood were excluded as the source of the organism. Contaminated tincture of thimerosal used to swab the rubber stoppers of blood culture bottles prior to venting (aerobic) or during blind subculturing after 24 hours of incubation (anaerobic) in the clinical microbiology laboratory was determined to be the cause of the pseudobacteremia. After appropriate safe-guards were implemented, we have continued to use tincture of thimerosal for these procedures with no further growth of C sordellii from blood cultures. The importance of less-conspicuous steps in the routine processing of culture material have been reemphasized.

Legionnaires' disease associated with rash and renal failure.

Legionnaires' disease is recognized as a multisystemic illness. Afflicted patients may have pulmonary, gastrointestinal tract, and central nervous system complications. However, dermal involvement is not well documented and renal insufficiency is uncommon and usually of mild severity. We report two consecutive cases of proven Legionella infection that were unusual in that a macular rash and profound renal failure requiring hemodialysis were noted. Skin biopsy specimens of the rash and autopsy findings suggest that these atypical features may have been mediated by the Legionella infection. Although it is not entirely clear from these two cases, we suggest that the skin and renal involvement may have been mediated by either a "toxin" elaborated by the organism, an immunologic response of the host to the organism, or some other unidentified mechanism.

Chemical injury of the spinal cord of the rabbit after intracisternal injection of gentamicin: an ultrastructural study.

We previously reported that rabbits with quadriplegia, after a single intracisternal injection of gentamicin sulfate, have multiple minute lesions in the white matter of the upper cervical spinal cord and the lower medulla oblongata, most marked at the C2 segment, when light microscopy is used to study the histopathologic changes. Using electron microscopy in the present investigations, we have found that 9 hours after injection of gentamicin sulfate there was marked edema of fibrous astrocytes in the soma and processes of glia limitans. Ribosomes were disarranged. Mitochondria were swollen, the matrix density was increased and contained paracrystalline lattice structure, and the cristae were reduced in number. Oligodendrocytes displayed hypertrophy, with proliferation of smooth ER, ribosomes, microtubules, Golgi complexes, and lysosomes. A few axons were subsegmentally swollen due to axoplasmic edema. At 24 hours, the lesions became obvious by light microscopy. Astrocytes were more edematous and oligodendrocytes remained hypertrophic. Myelin sheaths were tumefied by dissociation of myelin lamellae. Axons were edematously swollen. Lysed axons were surrounded by lysed myelin sheaths. At 48 hours, numerous neuroaxonal end bulbs were formed at the lysed end. Wallerian degeneration was also evident. The data suggest that oligodendroglia actively react to detoxify gentamicin, astroglia become severely edematous but survive, and axons and myelin sheaths are lysed in reaction to the toxicity of gentamicin. The unique distribution of gentamicin lesions in the deeper white matter, with sparing of the overlying marginal myelinated fibers, seems to depend primarily upon the distribution of edematous astrocytes, which are most sensitive to this noxious chemical. Since gentamicin is apparently detoxified by oligodendroglia, the regenerative process of axons starts after a short period of axonal lysis.

Chemical injury of the spinal cord of the rabbit after intracisternal injection of gentamicin.

A single intracisternal injection of 0.4 ml of 1.25 and 2.5 percent gentamicin sulfate with preservative to healthy adult rabbits caused acute respiratory paralysis and severe seizure activity initially and paralysis of the limbs subsequently. In the white matter of the upper cervical spinal cord, multiple, minute, disseminated, spongy lesions were observed. They consisted of lysis of axis cylinders, edematous dilatation of myelin sheaths, and loss of astroglia and interfascicular oligodendroglia. Axonal end-bulbs formed at the periphery of the lesions. Clinically and morphologically, 0.025 and 0.25 percent gentamicin sulfate solution did not produce myelopathy. The spinal lesions were distributed differently from those of other chemical myelopathies in that they developed in the deeper white matter with sparing of marginal myelinated fibers. Circumscribed high concentrations and gentamicin, and vulnerability of myelinated axis cylinders and interfascicular oligondendroglia to gentamicin may be the main factors causing these lesions. When gentamicin sulfate without preservative was injected, neutrophil leukocyte infiltration occurred actively in the spongy lesions. In the cervical spinal ganglia some nerve cells underwent cytoplasmic vacuolation. In control animals a single intracisternal injection of saline or preservative did not result in the production of these lesions.

How should xanthine oxidase-generated superoxide yields be measured?

The rate of formation of superoxide measured by its reduction of tetranitromethane (TNM) and by its reduction of ferric cytochrome c (Fe(III) cc) are in excellent agreement when the superoxide is generated from a simple chemical precursor. In contrast, the rate of formation of superoxide generated in the reaction of xanthine oxidase with acetaldehyde is much higher (up to a factor of 6) when measured with TNM and compared with Fe(III) cc. It is shown that Fe(III) cc measures superoxide that has diffused from the enzyme, and that TNM probably scavenges all the dioxygen that is reduced by one electron by the enzyme. The TNM traps enzyme-bound superoxide in competition with the second-electron transfer and proton transfer, which normally yield hydrogen peroxide. The proton transfer is probably rate determining, k(p)

Pneumonitis and pulmonary abscess associated with Moraxella nonliquefaciens.

A 78-year-old man with pneumonitis and pulmonary abscess associated with Moraxella nonliquefaciens is presented. This organism was found by culture of both transtracheal aspirate and sputum. No previous reports have associated M nonliquefaciens with infection of the lower respiratory tract, although sinusitis and bronchitis have been reported. Possible predisposing factors in our patient included carcinoma of the larynx, as well as alcohol ingestion and cigarette smoking.

Clinical significance of serotype, biotype, and beta-lactamase production of respiratory isolates of Hemophilus influenzae.

The correlation between clinical significance and serotype, biotype, and beta-lactamase production of 101 respiratory isolates of Hemophilus influenza from nonbacteremic patients was determined. Six of 33 (18.2%) isolates from patients with definite or probable infection were serotypable; only two of 67 (3%) isolates from colonized patients were serotypable. Eight-seven and one-half per cent of the serotypable strains were biotypes I or IV; 78.8% of nonserotypable strains were biotypes II, III, or V. Biotype distribution among isolates from patients with definite or probable infection were similar to isolates from colonized patients. beta-lactamase production was not helpful in evaluating clinical significance. Nonserotypable H. influenzae is an important cause of nonbacteremic pneumonia in elderly men.

Clinical significance of citrobacter isolates.

Little is know about the clinical significance of isolating Citrobacter in the clinical laboratory. During a one-year period, 116 Citrobacter isolates were obtained from 77 patients with 83 suspected infectious episodes. The majority of the suspected infectious episodes involved the urinary tract (45%) or respiratory tract (41%). Citrobacter diversus was associated with 42% of the episodes, Citrobacter freundii with 29%, and Citrobacter species with 29%. In 42% of the suspected infectious episodes, the presence of Citrobacter was considered clinically significant; in the others, the significance of the Citrobacter isolates was indeterminate or considered to be commensal. Two thirds of the significant infections were hospital-acquired. Most patients (73%) from whom Citrobacter was cultured had underlying diseases or factors predisposing to infection. These data suggest that Citrobacter is a cause of significant opportunistic nosocomial infection in the hospital.