Activity of combinations of dapsone, rifampin, minocycline, clarithromycin, and sparfloxacin against M. leprae-infected mice.

In these studies we evaluated the activity of low levels of five antimicrobials against Mycobacterium leprae-infected mice when administered singly and in all possible two- and three-drug combinations. Antibiotics studied were: dapsone (D) 0.0001% in the diet, rifampin (R) 20 mg/kg by gavage once monthly, minocycline (M) 0.004% in the diet, clarithromycin (C) 0.001% in the diet, and sparfloxacin (S) 5 mg/kg by gavage five times weekly. Singly each agent was found bacteriostatic (D + R) or partially bactericidal (M, C, and S) but not fully bactericidal. All 10 two-drug regimens were found at least bacteriostatic, 2 being "partially bactericidal" and 4 being "fully bactericidal." Of the 10 three-drug regimens, 9 were found "fully bactericidal" and the other "partially bactericidal." We conclude that combinations of antibiotics active against M. leprae are generally additive in combination.

Efficacy of minocycline in single dose and at 100 mg twice daily for lepromatous leprosy.

A clinical trial of minocycline in a total of 10 patients with previously untreated lepromatous leprosy was conducted in order to evaluate the efficacy of a single, initial, 200-mg dose and 100 mg twice daily of minocycline for a total duration of up to 3 months. Patients improved remarkably quickly. Although single-dose therapy did not result in a significant killing of Mycobacterium leprae, viable M. leprae were cleared from the dermis regularly by 3 months of twice-daily therapy, a rate similar to that achieved by minocycline 100 mg once daily. Because more side effects were noted herein than previously with 100 mg daily, we recommend that minocycline, when applied, be administered at 100 mg daily to leprosy patients.

Vaccination with pure Mycobacterium leprae proteins inhibits M. leprae multiplication in mouse footpads.

In this study, we evaluated vaccination with a number of purified, as well as recombinant, Mycobacterium leprae proteins for protective efficacy in mice. BALB/c mice were immunized intradermally with various native somatic (purified) or recombinant M. leprae proteins and their synthetic polypeptides emulsified in Freund's incomplete adjuvant. The protective efficacy of these preparations was assessed by enumeration of bacilli in the footpads of mice challenged with viable M. leprae 1 to 2 months following immunization. Protection was afforded by the purified and recombinant 10-kDa M. leprae cytoplasmic heat shock protein, the recombinant cell wall-associated 65-kDa M. leprae heat shock protein, and to a lesser extent, the purified 28-kDa M. leprae cytoplasmic protein (superoxide dismutase). Vaccination with either the purified or recombinant 35-kDa M. leprae cell membrane protein, the synthetic 27-amino-acid N-terminal peptide of the 10-kDa protein, the recombinant 18-kDa M. leprae protein, or the purified 22-kDa cell membrane protein was ineffective. When the interval between immunization and challenge was increased to 6 months, the purified 10-kDa M. leprae protein and the recombinant 65-kDa M. leprae protein lost vaccine efficacy, while a sodium dodecyl sulfate-soluble protein fraction of the M. leprae cell wall (soluble proteins), as had been found previously, continued to protect, suggesting that multiple M. leprae protein epitopes are critical for solid vaccine protection.

Effective vaccination of mice against Mycobacterium leprae with density-gradient subfractions of soluble M. leprae proteins: clues to effective protein epitopes.

It had previously been discovered that intradermal mouse vaccination with a protein fraction of Mycobacterium leprae (called soluble proteins) in Freund's incomplete adjuvant (FIA) resulted in consistent and long-lived protection against M. leprae multiplication from subsequent viable footpad challenges. In this study certain density-gradient subfractions of this soluble protein, but not others, in FIA afforded vaccine protection. The results of this study suggest which M. leprae proteins may be involved in protective immunity, particularly 1-3 kD, 10 kD, 65 kD, and those of higher molecular weight.

Reconstitution of Mycobacterium leprae immunity in severe combined immunodeficient mice using a T-cell line.

To test whether Mycobacterium leprae-immune T cells can confer protection against infection with leprosy bacilli, severe combined immunodeficient (SCID) mice were reconstituted with a BALB/c-derived, M. leprae-responsive, T-cell line. Flow cytometric analysis of spleen and peripheral blood cells confirmed reconstitution with T cells. In vitro lymphokine production and the proliferation of spleen cells from the reconstituted animals established that the donor cells had maintained their functional activity for the duration of the study (275 days). The transfer of immune T cells 24 hr before foot pad infection with leprosy bacilli resulted in a profound reduction in M. leprae multiplication, as compared to the nonreconstituted SCID mice. The yield of acid-fast bacilli in the foot pads of SCID mice reconstituted with the M. leprae-immune T cells also was significantly lower than that found in naive BALB/c mice, and at levels previously found only in BALB/c mice that had been immunized effectively. These experiments demonstrate that M. leprae-immune T cells home effectively and control M. leprae infection in SCID mice.

Dynamics of the quaternary conformational change in trout hemoglobin.

The kinetics of conformational changes in trout hemoglobin I have been characterized over the temperature range 2-65 degrees C from time-resolved absorption spectra measured following photodissociation of the carbon monoxide complex. Changes in the spectra of the deoxyheme photoproduct were used to monitor changes in the protein conformation. Although the deoxyheme spectral changes are only about 8% of the total spectral change due to ligand rebinding, a combination of high-precision measurements and singular value decomposition of the data permits a detailed analysis of both their amplitudes and relaxation rates. Systematic variation of the degree of photolysis was used to alter the distribution of liganded tetramers, permitting the assignment of the spectral relaxation at 20 microseconds to the R----T quaternary conformational change of the zero-liganded and singly liganded molecules and spectral relaxations at about 50 ns and 2 microseconds to tertiary conformational changes within the R structure. Analysis of the effect of photoselection by the linearly polarized excitation pulse indicates that a major contribution to the apparent geminate rebinding in the 50-ns relaxation arises from rotational diffusion of molecules containing unphotolyzed heme-CO complexes. The activation enthalpy and activation entropy for the R0----T0 transition are +7.4 kcal/mol and -12 cal mol-1 K-1. Using the equilibrium data, delta H = +29.4 kcal/mol and delta S = +84.4 cal mol-1 K-1 [Barisas, B. G., & Gill, S. J. (1979) Biophys. Chem. 9, 235-244], the activation parameters for the T0----R0 transition are calculated to be delta H = +37 kcal/mol and delta S = +73 cal mol-1 K-1. The similarity of the equilibrium and activation parameters for the T0----R0 transition indicates that the transition state is much more R-like than T-like. This result suggests that in the path from T0 to R0 the subunits have already almost completely rearranged into the R configuration when the transition state is reached, while in the path from R0 to T0 the subunits remain in a configuration close to R in the transition state. The finding of an R-like transition state explains why the binding of ligands causes much smaller changes in the R----T rates than in the T----R rates.

Effect of low-level and intermittent minocycline therapy on the growth of Mycobacterium leprae in mice.

We evaluated the minimal concentrations of minocycline in the diet and in serum required to inhibit the growth of seven Mycobacterium leprae isolates in mice. Minocycline concentrations of 0.01 and 0.04% in the diet, which resulted in levels in serum of less than or equal to 0.17 and 0.51 microgram/ml, respectively, were consistently and completely inhibitory. Even 0.004% dietary minocycline (levels in serum, less than or equal to 0.08 microgram/ml) partially inhibited five of these strains, while 0.001% minocycline was consistently inactive. For five of these isolates, minocycline at a concentration of 0.04% in the diet given 3 days (Monday, Wednesday, Friday) and 1 day weekly completely inhibited the growth of M. leprae, and minocycline given even 1 day monthly was partially inhibitory for three of these five M. leprae isolates.

Activities of various macrolide antibiotics against Mycobacterium leprae infection in mice.

We evaluated the activities of several macrolide antibiotics against M. leprae infections in mouse footpads. Erythromycin and azithromycin were inactive, while both roxithromycin and clarithromycin were found to be consistently active and, in fact, bactericidal. By both methods, clarithromycin was found to be superior to roxithromycin, a finding which, at least in part, may be a consequence of the higher levels of clarithromycin at the site of infection.

Primary dapsone-resistant Hansen's disease in California. Experience with over 100 Mycobacterium leprae isolates.

We found that in the years 1978 through 1981 only one of 54 previously untreated patients with Hansen's disease was found to harbor dapsone-resistant Mycobacterium leprae. That single strain was only partially resistant, ie, it was resistant to 0.0001% dapsone in a mouse diet but not to higher concentrations. During the years 1983 through 1988, M leprae from 47 previously untreated patients presenting to clinics in San Francisco, Calif, and Los Angeles, Calif, grew in mice. None of these strains was found to be dapsone resistant. Thus, from 1978 through 1988 only one of 101 M leprae isolates obtained from skin biopsy specimens from patients with leprosy was found to be resistant to dapsone. We have concluded that primary dapsone resistance still does not appear to be a significant problem in California. Owing to the fact that our single resistant case and those reported from international sources are, in general, partially resistant, the potential importance of partial dapsone resistance is discussed.

Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae.

Model vaccines against leprosy bacilli have consisted of nonvirulent, live, attenuated Mycobacterium bovis BCG and irradiated, heat-killed, or autoclaved intact M. leprae. We report that immunization with various cell wall fractions of M. leprae, progressively depleted of lipids, carbohydrates, and soluble proteins, as well as a partially purified protein(s) derived from a pellet fraction of sonicated M. leprae, conferred significant protection against subsequent infection with live leprosy bacilli. Moreover, lymphocytes from regional lymph nodes and spleens of mice immunized with these M. leprae-derived subunits responded by proliferation when stimulated with M. leprae in vitro. Our results provide the first evidence that vaccination with M. leprae-derived fractions protects mice against leprosy bacilli.

The effect of quaternary structure on the kinetics of conformational changes and nanosecond geminate rebinding of carbon monoxide to hemoglobin.

To determine the effect of quaternary structure on the individual kinetic steps in the binding of carbon monoxide to the alpha subunit of hemoglobin, time-resolved absorption spectra were measured after photodissociation of carbon monoxide from a hemoglobin tetramer in which cobalt was substituted for iron in the beta subunits. Cobalt porphyrins do not bind carbon monoxide. Spectra were measured in the Soret region at room temperature after time delays that varied from a few nanoseconds to the completion of ligand rebinding at about 100 ms. The results show that the liganded molecule, alpha(Fe-CO)2 beta(Co)2, is in the R state, but can be almost completely switched into the T state by the allosteric effectors inositol hexaphosphate and bezafibrate. The geminate yield, which is the probability that the ligand rebinds to the heme from within the protein, is found to be 40% for the R state and less than 1% for the T state. According to the simplest kinetic model, these results indicate that carbon monoxide enters the protein in the R and T quaternary conformations at the same rate, and that the 60-fold decrease in the overall binding rate, of carbon monoxide to the alpha subunit in the T state compared to the R state is almost completely accounted for by the decreased probability of binding after the ligand has entered the protein. The results further suggest that the low probability for the T state results from a decreased binding rate to the heme and not from an increased rate of return of the ligand to the solvent.

The nature of the primary photochemical events in rhodopsin and isorhodopsin.

The nature of the primary photochemical events in rhodopsin and isorhodopsin is studied by using low temperature actinometry, low temperature absorption spectroscopy, and intermediate neglect of differential overlap including partial single and double configuration interaction (INDO-PSDCI) molecular orbital theory. The principal goal is a better understanding of how the protein binding site influences the energetic, photochemical, and spectroscopic properties of the bound chromophore. Absolute quantum yields for the isorhodopsin (I) to bathorhodopsin (B) phototransformation are assigned at 77 K by using the rhodopsin (R) to bathorhodopsin phototransformation as an internal standard (phi R----B = 0.67). In contrast to rhodopsin photochemistry, isorhodopsin displays a wavelength dependent quantum yield for photochemical generation of bathorhodopsin at 77 K. Measurements at seven wavelengths yielded values ranging from a low of 0.089 +/- 0.021 at 565 nm to a high of 0.168 +/- 0.012 at 440 nm. An analysis of these data based on a variety of kinetic models suggests that the I----B phototransformation encounters a small activation barrier (approximately 0.2 kcal mol-1) associated with the 9-cis----9-trans excited-state torsional-potential surface. The 9-cis retinal chromophore in solution (EPA, 77 K) has the smallest oscillator strength relative to the other isomers: 1.17 (all-trans), 0.98 (9-cis), 1.04 (11-cis), and 1.06 (13-cis). The effect of conformation is quite different for the opsin-bound chromophores. The oscillator strength of the lambda max absorption band of I is observed to be anomalously large (1.11) relative to the lambda max absorption bands of R (0.98) and B (1.07). The wavelength-dependent photoisomerization quantum yields and the anomalous oscillator strength associated with isorhodopsin provide important information on the nature of the opsin binding site. Various models of the binding site were tested by using INDO-PSDCI molecular orbital theory to predict the oscillator strengths of R, B, and I and to calculate the barriers and energy storage associated with the photochemistry of R and I for each model. Our experimental and theoretical investigation leads to the following conclusions: (a) The counterion (abbreviated as CTN) is not intimately associated with the imine proton in R, B, or I. The counterion lies underneath the plane of the chromophore in R and I, and the primary chromophore-counterion electrostatic interactions involve C15-CTN and C13-CTN. These interactions are responsible for the anomalous oscillator strength of I relative to R and B. (b) The presence of a small activation barrier (~0.2 kcal mol-1) in the 9-cis - 9-trans excited-state surface is associated with the location of the counterion as well as the intrinsic photophysical properties of the 9-cis chromophore. The principal difference between the 1 1-cis -c 1 -transphoto reaction surface and the 9-cis - 9-trans photoreaction surface is the lack of effective electrostatic stabilization of distorted 9 = 10 conformations due to incomplete charge polarization. (c) Hydrogen bonding to the imine proton, ifpresent, does not involve the counterion. We conclude that water in the active site, or secondary interactions with the protein (not involving the CTN), are responsible. (d) All photochemical transformations involve one-bond photoisomerizations.This prediction is based on the observation of a very small excited state barrier for the I -- B photoreaction and a negative barrier for the R - B phototransformation, coupled with the theoretical prediction that all two-bond photoisomerizations have significant S, barriers while one-bond photoisomerizations have small to negative S, barriers.(e) Rhodopsin is energetically stabilized relative to isorhodopsin due to both electrostatic interactions and conformational distortion, both favoring stabilization of R. The INDO-PSDCI calculations suggest that rhodopsin chromophore-CTN electrostatic interactions provide an enhanced stabilization of -2 kcal mol-1 relative to I. Conformational distortion of the 9-cis chromophore-lysine system accounts for -3 kcal mol-1. (f) Energy storage in bathorhodopsin is-60% conformational distortion and 40% charge separation. Our model predicts that the majority of the chromophore protein conformational distortion energy involves interaction of the C,3(-CH3)=CI4--C,5=N-lysine moiety with nearby (unknown) protein residues. (g) Strong interactions between the counterion and the chromophore in R and I will generate weak, but potentially observable charge-transfer bands in the near infrared. The key predictions are the presence of an observable charge-transfer transition at 859 nm (1 1,640 cm- 1) in I and an analogous, but slightly weaker band at 897 nm (11,150 cm-1) in R. Both transitions involve the transfer of an electron from the counterion into low-lying l theta* molecular orbitals.

Time-resolved optical spectroscopy and structural dynamics following photodissociation of carbonmonoxyhemoglobin.

A summary is presented of our current understanding of the kinetics of ligand rebinding and conformational changes at room temperature following photodissociation of the carbon monoxide complex of hemoglobin with pulsed lasers. The events which occur subsequent to excitation have been followed over 12 decades in time, from about 100 fs to the completion of ligand rebinding at about 100 ms. Experiments with picosecond and subpicosecond lasers by others, together with molecular dynamics simulations, indicate that by 1 ns the deoxyhemoglobin photoproduct is in a thermally equilibrated ground electronic state, so that subsequent processes are unaffected by the initial laser excitation. The principal results have been obtained from time-resolved optical absorption spectroscopy using a sensitive nanosecond laser spectrometer. Five relaxations have been observed which are interpreted as geminate rebinding at about 50 ns that competes with motion of the ligand away from the heme which produces a tertiary conformational change, a second tertiary conformational change at 0.5-1 microseconds, transition from the R to T quaternary structure at about 20 microseconds, and overall bimolecular rebinding of ligands from the solvent to the R and T quaternary structures at about 200 microseconds and 10 ms. Assuming that the dissociation pathway in photolysis experiments is the reverse of the association pathway, we find that for the R state there is a 40% probability that the ligand will bind to the heme after entering the protein, and a 60% probability that it will return to the solvent. Studies on the alpha-subunit of an iron-cobalt hybrid hemoglobin indicate that carbon monoxide enters the protein at the same rate for both R and T quaternary structures. For the alpha-subunit in the T state the probability of binding after entry is much lower, and the ligand returns to the solvent more than 99% of the time, accounting for the 60-fold overall lower association rate. This decreased probability of binding results from a decreased rate of binding to the heme from within the protein, and not an increased rate of return to the solvent. There are still unresolved problems on the basic structural description of carbon monoxide binding and dissociation, particularly the functional significance of the tertiary relations in both the R and T states, and the precise number of kinetic barriers within the protein.

Energy storage in the primary photochemical events of rhodopsin and isorhodopsin.

The energetics associated with the photoequilibrium (Formula: see text) are measured at 77 K by using pulsed-laser photocalorimetry and a range of excitation wavelengths and relative starting concentrations. Enthalpies for the photochemical transformations R hv----B and I hv----B are measured to be delta HRB = 32.2 +/- 0.9 kcal mol-1 and delta HIB = 27.1 +/- 3.2 kcal mol-1, respectively. Although the value of delta HRB is slightly lower than that reported previously by Cooper of 34.7 +/- 2.2 kcal mol-1 [Cooper, A. (1979) Nature (London) 282, 531-533], the two values are in agreement within experimental error. The energy difference delta HRB - delta HIB = 5.1 +/- 3.3 kcal mol-1 is identical within experimental error with the difference in enthalpies of isorhodopsin and rhodopsin [5.2 +/- 2.3; Cooper, A. (1979) FEBS Lett. 100, 382-384]. We suggest that this result is consistent with the theory that bathorhodopsin is a single, common photochemical intermediate connecting rhodopsin and isorhodopsin.

Two-photon spectroscopy of locked-11-cis-rhodopsin: evidence for a protonated Schiff base in a neutral protein binding site.

We studied the nature of the protein binding site of rhodopsin, using two-photon spectroscopy to assign the location of the low-lying "covalent" 1Ag*- -like pi pi * state in a model rhodopsin containing a locked-11-cis chromophore. The two-photon thermal lens maximum is observed at 22,800 cm-1, approximately equal to 2000 cm-1 above the one-photon absorption maximum, indicating that the protein environment has induced a level ordering reversal of the low-lying pi pi * states relative to that observed in retinyl Schiff bases in solution. The spectroscopic results clearly indicate that the chromophore is protonated and that the binding site is uncharged. Electrostatic energy contour maps of the binding site are calculated, showing possible locations for the external counterion(s). Two models of the binding site are proposed that accommodate the available spectroscopic data. One model involves a protonated Schiff base chromophore stabilized by a single negatively charged aspartic or glutamic acid residue. A more complicated model involving two residues (one charged, the other neutral) is also proposed. The latter model is interesting because it also accommodates the observed deuterium isotope effect in the form of a proton translocation between the two residues. The translocation is assumed to be a ground state process, initiated subsequent to the photoisomerization of the chromophore and energetically driven via destabilization of the counterion environment as a result of isomerization-induced charge separation.