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The dynamics of a slender, nano-particle laden droplet are examined when it is subjected to an electric field. Under a long-wave assumption, the governing equations are reduced to a coupled pair of nonlinear evolution equations prescribing the dynamics of the interface and the depth-averaged particle concentration. This incorporates the effects of viscous stress, capillarity, electrostatically- induced Maxwell stress, van der Waals forces, evaporation and concentration-dependent rheology. It has previously been shown27that electric fields can be used to suppress the ring effect typically exhibited when such a droplet undergoes evaporation. We demonstrate here that the use of electric fields affords many diverse ways of controlling the droplets.

To review the computed tomography (CT) imaging features of pulmonary nocardiosis (PN) at the time of initial presentation.

Cytomegalovirus (CMV) is a significant cause of morbidity, mortality, and cost in solid organ transplant recipients. This study was conducted to measure both the clinical efficacy and the pharmacoeconomic impact of implementing, as standard of care, an abbreviated preemptive monitoring strategy compared with universal prophylaxis in a large teaching hospital.

ABSTRACTThe steady-state pharmacokinetics of lamivudine were evaluated in 11 subjects with human immunodeficiency virus infection and end-stage renal disease, 9 of whom were receiving hemodialysis and 2 of whom were receiving chronic ambulatory peritoneal dialysis (CAPD). All subjects received 150 mg of lamivudine daily for at least 2 weeks prior to sampling for determination of the pharmacokinetics of lamivudine over a 24-h period on 2 consecutive days. On the first day, subjects received 150 mg of oral lamivudine and underwent dialysis (hemodialysis or CAPD). On the second day, subjects received another 150 mg of oral lamivudine but dialysis was not performed. For the subjects undergoing hemodialysis, the geometric mean predose serum lamivudine concentration was 1.14 μg/ml (95% confidence interval [CI], 0.83 to 1.58 μg/ml), the geometric mean maximum concentration in serum (Cmax) was 3.77 μg/ml (95% CI, 3.01 to 4.71 μg/ml), and the geometric mean area under the serum concentration-time curve from time zero to 24 h (AUC0-24) was 49.8 μg · h/ml (95% CI 39.1 to 63.6 μg · h/ml). Hemodialysis removed approximately 28 mg of lamivudine but had no significant effect on Cmaxor AUC0-24. In the absence of hemodialysis, the geometric mean lamivudine terminal elimination half-life was 17.2 h (95% CI, 10.5 to 28.1 h), whereas the geometric mean intradialysis half-life of lamivudine was 5.3 h (95% CI, 3.4 to 8.2 h). The pharmacokinetics of lamivudine in subjects undergoing CAPD were similar to those in subjects undergoing hemodialysis. CAPD removed 24 mg of lamivudine over a 24-h period but had no effect on Cmaxor AUC0-24. Pharmacokinetic modeling suggests that a lamivudine dose of 25 mg daily in hemodialysis subjects would provide serum exposure similar to that provided by a dose of 150 mg twice daily in patients with normal renal function.

It is not easy to find candidate sites within a given protein where the geometry of the polypeptide chain matches that of metal-binding sites in known protein structures. By choosing a location in T4 lysozyme that is inherently flexible, it was possible to engineer a two-histidine site that binds different divalent cations. Crystallographic analysis shows that the geometry of binding of zinc is distorted tetrahedral while that of cobalt and nickel is octahedral. Insofar as spectroscopic data can be measured, they indicate that similar modes of coordination are retained in solution. The two substitutions, Thr21 → His and Thr142 → His, lie, respectively, on the surface of the N- and C-terminal domains on opposite sides of the active site cleft. The design takes advantage of hinge-bending motion which allows the binding site to adapt to the most favorable ligand geometry for the metal. Introduction of the two histidines increases the melting temperature of the protein by 2.0°C at pH 7.4. Metal binding further increases the melting temperature, but only by a small amount (up to 1.5°C). A third substitution, Gln141 → His, which could act as a third ligand in principle, does not do so, demonstrating the difficulty in mimicking naturally occurring metal-binding sites.

The mutation Glu108 → Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99 → Gly (L99G). Surprisingly, the two sites are 11 Å apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99 → Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 °C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20–30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 Å3. Instead of enlarging this cavity, mutant L99G results in a 4–5 Å displacement of part of helix F (residues 108–113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 Å3. Whether the mutation Glu108 → Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.

Zinc metallopeptidases that contain the His‐Glu‐Xaa‐Xaa‐His (HEXXH) motif generally have a third ligand of the metal ion that may be either a Glu residue (in clan MA) or a His residue (in clan MB) (Rawlings and Barrett (1995) Methods Enzymol. 248, 183–228). Thimet oligopeptidase has not yet been assigned to either clan, and both Glu and His residues have been proposed as the third ligand. We mutated candidate ligand residues in the recombinant enzyme and identified Glu, His and Asp residues that are important for catalytic activity and/or stability of the protein. However, neither of the Glu and His residues close to the HEXXH motif that have previously been suggested to be ligands is required for the binding of zinc. We conclude that thimet oligopeptidase is not a member of clan MA or clan MB and it is likely that the enzyme possesses a catalytic site and protein fold different from those identified in any metallopeptidase to date. The definitive identification of the third zinc ligand may well require the determination of the crystallographic structure of thimet oligopeptidase or one of its homologues.

1. Purified botulinum neurotoxin type A (10 nM) was applied in vitro to mouse diaphragm muscles. Intracellular micro‐electrode recordings were made continuously in single fibres. 2. This treatment reduced end‐plate potential (e.p.p.) amplitudes with a time to half‐maximal effect of about 75 min at 22‐25 degrees C. E.p.p. rise‐times remained fast and unaffected by the toxin. 3. Miniature end‐plate potential (m.e.p.p.) frequency was reduced by the toxin to less than 5% of control frequency, and followed a similar time course to the block of e.p.p. amplitudes. The m.e.p.p. rise‐time and coefficient of variation (c.v.) of m.e.p.p. amplitude distributions both increased, but the time course of these increases lagged significantly behind the change in frequency. 4. A population of slow rise‐time m.e.p.p.s was present in controls at low frequency. This population was found to be unaffected by the toxin. 5. The above‐detailed in vitro changes could be explained by the toxin acting by a single common mechanism to inhibit the release process underlying both fast rise‐time m.e.p.p.s and e.p.p.s. A distinct release process, which leads to slow rise‐time m.e.p.p.s, was unaffected by the toxin.

1. The action of immunoglobulin G obtained from patients with Lambert‐Eaton myasthenic syndrome (LEMS IgG) was investigated by injecting mice, followed by intracellular recordings from the mouse diaphragm. 2. End‐plate potential quantal content was studied over a range of Ca2+ concentrations. Curves of log quantal content versus log Ca2+ concentration were shifted to the right by LEMS IgG. For low Ca2+ concentrations, release continued to follow Poisson statistics after LEMS IgG treatment. 3. Miniature end‐plate potential (m.e.p.p.) frequency was measured in solutions containing high K+ concentrations. LEMS IgG significantly reduced m.e.p.p. frequency at each K+ concentration studied. 4. M.e.p.p. frequency was measured at fixed high‐K+ concentration (15.9 mM) for a range of Ca2+ concentrations. The log‐log plot of m.e.p.p. frequency versus Ca2+ concentration was shifted downwards throughout by LEMS IgG. 5. M.e.p.p. frequency was not affected by LEMS IgG in Ca2+‐free solutions (K+ concentration 15.9 mM) or in solutions of low Ca2+ concentration (K+ concentration 5.9 mM). 6. At each Ca2+ concentration studied, m.e.p.p. amplitudes were not affected by LEMS IgG. 7. The data suggest that LEMS IgG acts on presynaptic voltage‐dependent Ca2+ channels to cause their loss of function, probably by down‐regulation.

1. The effect of Lambert‐Eaton myasthenic syndrome (LEMS) immunoglobulin G (IgG) on Ca2+ channels in undifferentiated mouse neuroblastoma x rat glioma hybrid cells (NG 108 15) was studied using the whole‐cell patch clamp technique. 2. Sustained inward Ca2+ channel currents were evoked by depolarizing pulses from holding potentials of ‐80 and ‐40 mV, and were blocked by 5 microM‐nitrendipine (L‐type currents). Transient inward Ca2+ channel currents were activated from a holding potential of ‐80 mV by small depolarizing steps (T‐type currents). Noradrenaline (10 microM) was without effect on transient currents. 3. LEMS IgG selectively reduced sustained (L‐type) Ca2+ channel current amplitudes evoked from either holding potential used. In the presence of nitrendipine (5 microM), there was no significant effect of LEMS IgG on the remaining transient (T‐type) Ca2+ channel current amplitudes. 4. Studies of the potential for maximal inward current indicated that voltage sensitivities of both L‐ and T‐type Ca2+ channel current amplitudes were unaffected by LEMS IgG, whether recorded in the presence or absence of nitrendipine. LEMS IgG had no significant effect on the time‐to‐peak or decay of Ca2+ channel currents. 5. It is concluded that LEMS IgG acts selectively to cause functional loss of L‐type, but not T‐type, Ca2+ channels in NG 108 15 cells. Any effect of LEMS IgG on N‐type channels (not present in these undifferentiated cells) was not studied here. LEMS IgG also acts at motor nerve terminal Ca2+ channels leading to muscle weakness. Thus antigenic similarities must exist between L‐type channels in NG 108 15 cells and Ca2+ channels at motor nerve terminals.

1. The acute effects of antibodies (both polyclonal and monoclonal) raised against the acetylcholine receptor were studied at mouse and rat end‐plates. Isolated muscles were incubated in solutions containing antibody for 2 1/4 to 3 1/2 h. Intracellular microelectrode techniques were then used to record miniature end‐plate potentials (MEPPs) and voltage noise. 2. Most antibody preparations investigated did not reduce MEPP amplitudes as compared with controls. One monoclonal (C7) and one polyclonal (J) preparation irreversibly reduced MEPP amplitudes. Both preparations caused reductions in acetylcholine‐induced depolarization and associated channel opening frequency (from voltage noise analysis). Single‐channel depolarization was not altered by these antibodies. 3. On the basis of these and previous results, four antibody binding regions on the receptor surface were distinguished according to whether channel function and/or alpha‐bungarotoxin binding were affected. Although most antibody preparations did not affect channel function, monoclonal antibody C7 appeared to alter function by acting on the channel itself so as to prevent channel opening.

The 5-methylthio-D-ribose moiety of 5′-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 1,2-Dihydroxy-3-keto-5-methylthiopentene anion (an aci-reductone) is an advanced intermediate in the methionine salvage pathway present in the Gram-negative bacterium Klebsiella pneumoniaeand rat liver. This metabolite is oxidized spontaneously in air to formate and 2-keto-4-methylthiobutyric acid (the α-keto acid precursor of methionine). Previously, we had purified an enzyme (E2) from Klebsiellawhich catalyzes the oxidative degradation of the aci-reductone to formate, CO, and methylthiopropionic acid. To further characterize the reactions of the aci-reductone we used its desthio analog, 1-2-dihydroxy-3-ketohexene anion (III), which was described previously. This molecule undergoes the analogous enzymatic and non-enzymatic reactions of the natural substrate, namely the formation of formate, CO, and butyrate from III. Experiments with 18O2show that E2 is a dioxygenase which incorporates one molecule of 18O into formate and butyric acid. No cofactor has been identified. We were unable to find an enzyme which catalyzes the conversion of 1,2-dihydroxy-3-keto-5-methylthiopentane to a keto acid precursor of methionine. The keto acid is probably produced non-enzymically in Klebsiella.We have, however, identified and purified an enzyme (E3) from rat liver, which catalyzes the formation of formate and 2-oxopentanoic acid from III. This enzyme has a monomeric molecular mass of 28,000 daltons, and no chromophoric cofactor has been identified. Experiments with 18O2show that E3 is a dioxygenase which incorporates an 18O molecule into formate and the α-keto acid. In rat liver CO formation was not detected.

The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 2,3-Diketo-5-methylthio-1-phosphopentane is an advanced intermediate in the methionine recycling pathway present in the Gram-negative bacterium Klebsiella pneumoniae. This unusual metabolite is oxidatively cleaved to yield formate (from C-1), 2-keto-4-methylthiobutyrate (the transamination product of methionine), and 3-methylthiopropionate. To further characterize this oxidative conversion, the desthio analog of the naturally occurring diketone, namely 2,3-diketo-1-phosphohexane I, was synthesized. If the metabolism of I is analogous to that of 2,3-diketo-5-methylthio-1-phosphopentane it should be converted to formate, 2-ketopentanoate, and butyrate. An enzyme (E-1), which mediates the oxidative conversion of I to formate and 2-ketopentanoate, was isolated from extracts of K. pneumoniae. E-1 was purified 100-fold to homogeneity in 10% yield. The native enzyme is a monomeric protein of M(r) 27,000. The activity of E-1 requires magnesium ion as a cofactor. No other prosthetic groups were detected. Incubation of the enzyme with I, under anaerobic conditions, led to the discovery of two intermediates. These species have been identified by 1H and 13C NMR, UV-visible spectroscopy, and model chemistry studies as 2-hydroxy-3-keto-1-phospho-1-hexene II, generated by enolization of I; and 1,2-dihydroxy-3-keto-1-hexene III, generated by enzymatic dephosphorylation of II. Intermediates II and III are released from the active site of the enzyme; III accumulates under anaerobic conditions. Under aerobic conditions, III is non-enzymically oxidized to 2-ketopentanoate, formate, and other products. Compound II was also generated by heating I at pH 7.5 for 7 min. Action of alkaline phosphatase on II produces III.

Exposure to MHC antigens by kidney transplantation can result in long-term sensitization or tolerance. In order to characterize immune responses to an acutely rejected renal allograft, ACI (RT1a) kidneys were transplanted into untreated male PVG (RT1e) recipients and allowed to reject while one native kidney remained in situ for host survival. Two distinct patterns of recipient sensitization were found based on early IgM responses to RT1.Aafollowing rejection, with “weakly sensitized” and “strongly sensitized” groups comprising approximately 40 and 60, respectively, of the recipients. Serum taken from strongly versus weakly sensitized animals at the time of peak IgM responses (7 days post-transplantation) showed greater ability to block binding of anti-RT1.AamAb (R2/15S, R2/10P, or YR1/100) to PVG.1A (RT1aclass I and II on a PVG background) lymph node target cells. Sera obtained from strongly sensitized recipients during peak IgG responses (4 weeks after kidney transplantation) demonstrated significantly higher IgG2a and IgG2b alloantibody levels than weakly sensitized rats at all serum dilutions (1:4–1:1024). Allografts harvested 10 days after transplantation from strongly sensitized recipients (strong R2/10P-blocking serum, n=9) had a vascular pattern of rejection characterized primarily by extensive vascular endothelial damage, glomerular and cortical necrosis, and gross infarction of the graft. In contrast, grafts harvested from weakly sensitized recipients at 10 days, 21 days, or >6 months (n=6, 10, and 6, respectively) posttransplantation showed a significantly different pattern of rejection, with moderate interstitial lymphocytic infiltrates but substantial preservation of the general kidney architecture. When challenged 13 weeks posttransplantation, strongly sensitized animals rejected RT1.Aaclass I-disparate PVG.R1 skin grafts in an accelerated fashion, whereas such grafts survived indefinitely on weakly sensitized recipients. These findings indicate that two patterns of renal allograft rejection can occur between a fixed strain disparity, one of

The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 1,2-Dihydroxy-3-keto-5-methylthiopentene anion (an aci-reductone) is an advanced intermediate in the methionine salvage pathway present in the Gram-negative bacterium Klebsiella pneumoniae and rat liver. This metabolite is oxidized spontaneously in air to formate and 2-keto-4-methylthiobutyric acid (the alpha-keto acid precursor of methionine). Previously, we had purified an enzyme (E2) from Klebsiella which catalyzes the oxidative degradation of the aci-reductone to formate, CO, and methylthiopropionic acid. To further characterize the reactions of the aci-reductone we used its desthio analog, 1-2-dihydroxy-3-ketohexene anion (III), which was described previously. This molecule undergoes the analogous enzymatic and non-enzymatic reactions of the natural substrate, namely the formation of formate, CO, and butyrate from III. Experiments with 18O2 show that E2 is a dioxygenase which incorporates one molecule of 18O into formate and butyric acid. No cofactor has been identified. We were unable to find an enzyme which catalyzes the conversion of 1,2-dihydroxy-3-keto-5-methylthiopentane to a keto acid precursor of methionine. The keto acid is probably produced non-enzymically in Klebsiella. We have, however, identified and purified an enzyme (E3) from rat liver, which catalyzes the formation of formate and 2-oxopentanoic acid from III. This enzyme has a monomeric molecular mass of 28,000 daltons, and no chromophoric cofactor has been identified. Experiments with 18O2 show that E3 is a dioxygenase which incorporates an 18O molecule into formate and the alpha-keto acid. In rat liver CO formation was not detected.

Renal allograft rejection in rats and humans is a potent inducer of alloantibody to donor major histocompatibility complex antigens. Alloantibody in such presensitized recipients can cause hyperacute rejection of subsequent renal allografts. In order to characterize alloantibody production in rats presensitized by renal graft rejection, ACI (RT1a) kidneys were transplanted into untreated fully allogeneic PVG (RTlc) recipients and allowed to reject while one native kidney remained in situ for host survival. Serum samples collected at weekly intervals were analyzed by flow cytometry for IgM and IgG antibody binding to ACI lymphoid target cells. The specificity of alloantibody responses was assessed by (1) differential binding to congenic rat strain target cells expressing only donor class I (PVG.R1) versus both donor class I and II (PVG.1A) antigens, (2) differential binding to unseparated donor lymphoid target cells versus lymphoid target cells depleted of class II MHC antigen-expressing cells, and (3) specific blocking of monoclonal antibodies to donor class I (R2/10P, R2/15S) or class II (F17.23.2) epitopes. Alloantibody responses to both donor class I and II MHC antigens were detected. The initial IgM response to donor class I MHC antigens peaked at the time of rejection, followed by a steady decline to relatively low levels by 4 weeks posttransplantation. The IgM response to donor class II MHC antigens was found to be cyclical with apparent peaks at day 7 and 5–6 weeks. The IgG response to donor class I and class II MHC antigens reached maximum by 5–6 weeks before slowly decreasing. IgM and IgG alloantibody specific for class I and class II MHC antigens could be detected through 19 weeks posttransplantation.

Two patients are described with a myasthenic syndrome that presented in early adult life. One patient had 2 asymptomatic first degree relatives with similar electrophysiological findings. Both patients had abnormal fatiguability, arm weakness being prominent; neither of them responded to anti-cholinesterase medication. An abnormal decrement at 3 Hz stimulation was present, and a single stimulus evoked a repetitive response. Electrophysiological studies on biopsied intercostal muscle showed miniature endplate potentials of normal amplitudes but with prolonged rise and decay times. Anticholinesterase staining (Case 1) was not reduced, and showed elongation of some endplates. Ultrastructural studies (Case 2) showed degeneration of junctional folds and diffusely thickened endplate basal lamina. Calcium deposits were not observed and myopathic changes were slight. The findings are consistent with a prolonged open time of the ACh-induced ion channel.