Homoserine dehydrogenase from Saccharomyces cerevisiae: kinetic mechanism and stereochemistry of hydride transfer.

Homoserine dehydrogenase (HSD), which is required for the synthesis of threonine, isoleucine and methionine in fungi, is a potential target for novel antifungal drugs. In order to design effective inhibitors, the kinetic mechanism of Saccharomyces cerevisiae HSD and the stereochemistry of hydride transfer were examined. Product inhibition experiments revealed that yeast HSD follows an ordered Bi Bi kinetic mechanism, where NAD(P)H must bind the enzyme prior to aspartate semialdehyde (ASA) and homoserine is released first followed by NAD(P)+. H-(1,2,4-triazol-3-yl)-D,L-alanine was an uncompetitive inhibitor of HSD with respect to NADPH (K(ii)=3.04+/-0.18 mM) and a noncompetitive inhibitor with respect to ASA (K(is)=1.64+/-0.36 mM, K(ii)=3.84+/-0.46 mM), in agreement with the proposed substrate order. Both kinetic isotope and viscosity experiments provided evidence for a very rapid catalytic step and suggest nicotinamide release to be primarily rate limiting. Incubation of HSD with stereospecifically deuterated NADP[2H] and subsaturating amounts of aspartate semialdehyde revealed that the pro-S NADPH hydride is transferred to the aldehyde. The pH dependence of steady state kinetic parameters indicate that ionizable groups with basic pKs may be involved in substrate binding, consistent with the observation of Lys223 at the enzyme active site in the recently determined 3D structure [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. These findings provide the requisite foundation for future exploitation of fungal HSD in inhibitor design.

Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity.

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.

In vivo determination of optical properties of normal and tumor tissue with white light reflectance and an empirical light transport model during endoscopy.

Determination of tissue optical properties is fundamental for application of light in either therapeutical or diagnostics procedures. In the present work we implemented a spatially resolved steady-state diffuse reflectance method where only two fibers (one source and one detector) spaced 2.5 mm apart are used for the determination of the optical properties. The method relies on the spectral characteristics of the tissue chromophores (water, dry tissue, and blood) and the assumption of a simple wavelength dependent expression for the determination of the reduced scattering coefficient. Because of the probe dimensions the method is suited for endoscopic measurements. The method was validated against more traditional models, such as the diffusion theory combined with adding doubling for in vitro measurements of bovine muscle. Mean and standard deviation of the absorption coefficient and the reduced scattering coefficient at 630 nm for normal mucosa were 0.87+/-0.22 cm(-1) and 7.8+/-2.3 cm(-1), respectively. Cancerous mucosa had values 1.87+/-1.10 cm(-1) and 8.4+/-2.3 cm(-1), respectively. These values are similar to data presented by other authors. Blood perfusion was the main variable accounting for differences in the absorption coefficient between the studied tissues.

Circular polarization memory in polydisperse scattering media.

We investigate the survival of circularly polarized light in random scattering media. The surprising persistence of this form of polarization has a known dependence on the size and refractive index of scattering particles, however a general description regarding polydisperse media is lacking. Through analysis of Mie theory, we present a means of calculating the magnitude of circular polarization memory in complex media, with total generality in the distribution of particle sizes and refractive indices. Quantification of this memory effect enables an alternate pathway toward recovering particle size distribution, based on measurements of diffusing circularly polarized light.

Putative photoacoustic damage in skin induced by pulsed ArF excimer laser.

Argon-fluoride excimer laser ablation of guinea pig stratum corneum causes deeper tissue damage than expected for thermal or photochemical mechanisms, suggesting that photoacoustic waves have a role in tissue damage. Laser irradiation (193 nm, 14-ns pulse) at two different radiant exposures, 62 and 156 mJ/cm2 per pulse, was used to ablate the 15-microns-thick stratum corneum of the skin. Light and electron microscopy of immediate biopsies demonstrated damage to fibroblasts as deep as 88 and 220 microns, respectively, below the ablation site. These depths are far in excess of the optical penetration depth of 193-nm light (1/e depth = 1.5 micron). The damage is unlikely to be due to a photochemical mechanism because (a) the photons will not penetrate to these depths, (b) it is a long distance for toxic photoproducts to diffuse, and (c) damage is proportional to laser pulse intensity and not the total dose that accumulates in the residual tissue; therefore, reciprocity does not hold. Damage due to a thermal mechanism is not expected because there is not sufficient energy deposited in the tissue to cause significant heating at such depths. The damage is most likely due to a photoacoustic mechanism because (a) photoacoustic waves can propagate deep into tissue, (b) the depth of damage increases with increasing laser pulse intensity rather than with increasing total residual energy, and (c) the effects are immediate. These effects should be considered in the evaluation of short pulse, high peak power laser-tissue interactions.

Controlled removal of human stratum corneum by pulsed laser.

A new method is presented for controlled removal of the stratum corneum of human skin. An excimer laser (193 nm wavelength, 14 ns pulsewidth) was used to remove stratum corneum from in vitro human skin samples by an ablative process. The tritiated water (3H2O) permeability constant and electrical resistance of skin samples were measured in a diffusion chamber apparatus to quantify the enhancement of skin permeability. Each laser pulse ablates about a micrometer of stratum corneum, which allows controlled removal of tissue. The maximum specific enhancement of the 3H2O permeability constant obtained after complete stratum corneum removal depends on the laser pulse energy used. The most gentle laser ablation, achieved with a radiant exposure of 70 mJ/cm2 per pulse, produced a 124-fold enhancement, which is comparable to that achieved after stratum corneum removal by tape-stripping or removal of epidermis by mild heat treatment. Rapid tissue ablation occurred at higher radiant exposures of 170-480 mJ/cm2 per pulse, but only a 45-fold enhancement of permeability was achieved. The precision with which stratum corneum can be ablated using excimer laser pulses may allow further basic research on the internal structure of stratum corneum and on the re-epithelization in controlled wounds. The technique may prove useful clinically to enhance percutaneous transport in applications such as topical delivery of drugs, patch testing, and percutaneous blood gas monitoring.

The systemic administration of gamma interferon inhibits collagen synthesis and acute inflammation in a murine skin wounding model.

The ability of gamma interferon (IFN-gamma) to affect cutaneous collagen synthesis in vivo was examined in a murine wounding model. Reproducible areas of full-thickness skin necrosis were produced by argon laser radiation. Mice received recombinant murine IFN-gamma (rMuIFN-gamma) (8.7 X 10(3) units/hr) over 14 d via osmotic pumps implanted subcutaneously or intraperitoneally. At 14 and 21 d after wounding, there was less fibrous tissue in healing scars of treated animals as determined by light and transmission electron microscopy. Associated with the decrease in connective tissue was an increase in the acid mucopolysaccharide content of healing scars, which was largely hyaluronate. Quantitative image analysis of electron micrographs confirmed that less collagen was present in healing scars of animals receiving rMuIFN-gamma. The mean cross-sectional area of collagen fibers was smaller in specimens from treated mice, but no difference was seen in the size of collagen fibrils. The time required to obtain full skin closure was also delayed 23%-27% in treated animals. Using this injury model, we also found that rMuIFN-gamma significantly reduced the degree of perilesional erythema surrounding the laser injury sites and, in the first 6 d after wounding, the degree of polymorphonuclear infiltrate present histologically at lesional sites. Indeed, rMuIFN-gamma also decreased the cutaneous accumulation of neutrophils induced by known proinflammatory mediators, such as interleukin 1 and activated serum. Thus, systemically administered IFN-gamma not only down-regulates collagen synthesis in the skin but also modulates in a previously unrecognized manner: neutrophil accumulation at sites of tissue injury in vivo.

Melanosomes are a primary target of Q-switched ruby laser irradiation in guinea pig skin.

The specific targeting of melanosomes may allow for laser therapy of pigmented cutaneous lesions. The mechanism of selective destruction of pigmented cells by various lasers, however, has not been fully clarified. Black, brown, and albino guinea pigs were exposed to optical pulses at various radiant exposure doses from a Q-switched, 40 nsec, 694 nm ruby laser. Biopsies were analyzed by light and electron microscopy (EM). Albino animals failed to develop clinical or microscopic evidence of cutaneous injury after irradiation. In both black and brown animals, the clinical threshold for gross change was 0.4 J/cm2, which produced an ash-white spot. By light microscopy, alterations appeared at 0.3 J/cm2 and included separation at the dermoepidermal junction, and the formation of vacuolated epidermal cells with a peripheral cytoplasmic condensation of pigment. By EM, enlarged melanosomes with a central lucent zone were observed within affected epidermal cells at 0.3 J/cm2. At 0.8 and 1.2 J/cm2, individual melanosomes were more intensely damaged and disruption of melanosomes deep in the hair papillae was observed. Dermal-epidermal blisters were formed precisely at the lamina lucida, leaving basal cell membranes and hemidesmosomes intact. Possible mechanisms for melanosomal injury are discussed. These observations show that the effects of the Q-switched ruby laser are melanin-specific and melanin-dependent, and may be useful in the selective destruction of pigmented as well as superficial cutaneous lesions.

Mid-infrared laser ablation of stratum corneum enhances in vitro percutaneous transport of drugs.

The precise removal of stratum corneum from cadaveric swine skin by a mid-infrared erbium:yttrium scandium gallium garnet laser (lambda = 2.79 microns; 250 microseconds pulse width) was assessed by electrical resistance measurements and documented by histology. The effects of stratum corneum removal by laser ablation and by adhesive tape-stripping on the in vitro penetration of 3H-hydrocortisone and 125I-gamma-interferon were determined. Excised swine skin was irradiated with laser (1 J/cm2; 31 mJ/pulse; 1 Hz; 2 mm spot diameter). For skin penetration studies, laser pulses were delivered to discrete 2-mm areas to ablate up to 12.6% of the total 3-cm2 stratum corneum diffusional area. Franz in vitro skin penetration chambers were used to measure the cumulative 48-h penetration of 3H-hydrocortisone and 125I-gamma-interferon in laser-treated and tape-stripped skin. Electrical resistance measurements and histologic studies demonstrated that 10-14 laser pulses at the above energy density were required to abolish skin resistance and selectively ablate stratum corneum without damage to adjacent dermal structures. Laser ablation of 12.6% of the surface area of stratum corneum produced a 2.8 and 2.1-times increase in permeability constant (kp) for 3H-hydrocortisone and 125I-gamma-interferon, respectively. These studies demonstrate that a pulsed mid-infrared laser can reliably and precisely remove the stratum corneum, facilitating penetration of large molecules such as 125I-gamma-interferon that cannot penetrate intact skin. This new technique may be useful for basic and clinical investigation of skin barrier properties.

Path integral description of light transport in tissue.

The early photons that arrive at a collector through a large thickness of tissue have potential for imaging internal organ structure, function, and status with improved image resolution relative to late arriving photons which have been diffusely scattered. Imaging algorithms require a theory to calculate early photon arrival for comparison with experimental data. The path integral description of light transport describes the movement of photons as particles undergoing collisions in a scattering medium based on the Brownian motion formalism of Feynman and Hibbs (unconstrained path) which applies the principle of least action. Including the additional constraint that photons have a constant velocity of c yields paths that conserve the speed of light (constrained path). This paper outlines the basic derivation of the path integral method and compares the constrained and unconstrained paths.

Dynamic epidermal cooling during pulsed laser treatment of port-wine stain. A new methodology with preliminary clinical evaluation.

BACKGROUND AND DESIGN: The clinical objective in the treatment of a patient with port-wine stain (PWS) undergoing laser therapy is to maximize thermal damage to the PWS, while at the same time minimizing nonspecific injury to the normal overlying epidermis. With dynamic cooling, the epidermis can be cooled selectively. When a cryogen spurt is applied to the skin surface for an appropriately short period of time (on the order of tens of milliseconds), the cooling remains localized in the epidermis, while leaving the temperature of the deeper PWS vessels unchanged. RESULTS: Comparative measurements obtained by a fast infrared imaging detector demonstrated that the surface temperature prior to laser exposure could be reduced by as much as 40 degrees C using the dynamic cooling technique. No skin surface textural changes were noted on PWS test sites cooled with a 20- to 80-millisecond cryogen spurt after flashlamp-pumped pulsed dye laser (FLPPDL) exposure (lambda = 585 nm; tau p = 450 microseconds) at the maximum light dosage possible (10 J/cm2). In contrast, epidermal necrosis occurred on the uncooled sites after such exposure. Six months after laser exposure, clinically significant blanching on the cooled sites indicates laser photothermolysis of PWS blood vessels did occur. CONCLUSIONS: Our preliminary experiments demonstrate the feasibility of selectively cooling the normal overlying epidermis without affecting the temperature of the deeper PWS vessels. Furthermore, protection of the epidermis from thermal injury, produced by melanin light absorption at clinically relevant wavelengths, can be achieved effectively. An additional advantage of dynamic epidermal cooling is reduction of patient discomfort associated with FLPPDL therapy. Further studies are under way to determine an optimum strategy for applying this dynamic cooling technique during pulsed laser treatment of patients with PWS and others with selected dermatoses (dermal melanocytic lesions and tattoos).

Modeling photon transport in transabdominal fetal oximetry.

The possibility of optical oximetry of the blood in the fetal brain measured across the maternal abdomen just prior to birth is under investigation. Such measurements could detect fetal distress prior to birth and aid in the clinical decision regarding Cesarean section. This paper uses a perturbation method to model photon transport through an 8-cm-diam fetal brain located at a constant 2.5 cm below a curved maternal abdominal surface with an air/tissue boundary. In the simulation, a near-infrared light source delivers light to the abdomen and a detector is positioned up to 10 cm from the source along the arc of the abdominal surface. The light transport [W/cm2 fluence rate per W incident power] collected at the 10 cm position is Tm = 2.2 x 10(-6) cm(-2) if the fetal brain has the same optical properties as the mother and Tf = 1.0 x 10(-6) cm(-2) for an optically perturbing fetal brain with typical brain optical properties. The perturbation P=(Tf - Tm)/Tm is -53% due to the fetal brain. The model illustrates the challenge and feasibility of transabdominal oximetry of the fetal brain.

Optimized radial and angular positions in Monte Carlo modeling.

In Monte Carlo simulations of light transport in tissues, a grid system is set up to score physical quantities. This study of cylindrically symmetrical problems found that the optimized radial and angular positions for the averaged physical quantities in each grid element are off-center. The error of the extrapolated physical quantities at the light-incidence point using the centered radial positions is up to 14.3%.

Influence of blood vessels on the measurement of hemoglobin oxygenation as determined by time-resolved reflectance spectroscopy.

We report the development of a heterogeneous resin-tube model to study the influence of blood vessels on the apparent absorption of the system, mu a(sys), using a time-resolved technique. The experimental results show that mu a(sys) depends on the absorption inside the tubes, mu a(tube), tube diameters, and tube-to-sample volume ratios. A mathematical expression relating mu a(sys) and mu a(tube) is derived based on the experimental results and is verified by time-resolved Monte Carlo simulations for heterogeneous models. This analytical formula predicts that the apparent absorption coefficient measured on a biological organ is a volume-weighted sum of the absorption coefficients of different absorbing components. We present some apparent absorption coefficients measured in vivo in animals and humans and discuss improved algorithms that calculate the hemoglobin saturation by including background-tissue absorption and blood vessel distribution.

Light distributions from point, line and plane sources for photochemical reactions and fluorescence in turbid biological tissues.

Light distributions in biological tissues are summarized in simple expressions for spherical, cylindrical and planar geometries due to point sources, line sources and planar sources. The goal is to provide workable tools for computing light distributions that govern the amount and distribution of photochemical reactions in experimental solutions, films and biological tissues. Diffusion theory expressions are compared with Monte Carlo simulations. Analytic expressions that mimic accurate Monte Carlo simulations are presented. Application to fluorescence measurements and prediction of necrotic zones in photodynamic therapy are outlined.

The melanosome: threshold temperature for explosive vaporization and internal absorption coefficient during pulsed laser irradiation.

The explosive vaporization of melanosomes in situ in skin during pulsed laser irradiation (pulse duration less than 1 microsecond) is observed as a visible whitening of the superficial epidermal layer due to stratum corneum disruption. In this study, the ruby laser (694 nm) was used to determine the threshold radiant exposure, H0 (J/cm2), required to elicit whitening for in vitro black (Negroid) human skin samples which were pre-equilibrated at an initial temperature, Ti, of 0, 20, or 50 degrees C. A plot of H0 vs Ti yields a straight line whose x-intercept indicates the threshold temperature of explosive vaporization to be 112 +/- 7 degrees C (SD, N = 3). The slope, delta H0/delta Ti, specifies the internal absorption coefficient, mua, within the melanosome: mua = -rho C/(slope(1 + 7.1 Rd)), where rho C is the product of density and specific heat, and Rd is the total diffuse reflectance from the skin. A summary of the absorption spectrum (mua) for the melanosome interior (351-1064 nm) is presented based on H0 data from this study and the literature. The in vivo absorption spectrum (380-820 nm) for human epidermal melanin was measured by an optical fiber spectrophotometer and is compared with the melanosome spectrum.

Immediate pigment darkening: visual and reflectance spectrophotometric analysis of action spectrum.

Immediate pigment darkening (IPD) occurs in human skin upon exposure to ultraviolet-A and visible radiation. The spectral changes that occur during IPD were measured with a rapid scanning reflectance spectrophotometer (RS) which employs optical fiber bundles for delivery and detection of light between 400 and 750 nm. The radiation dose dependence and wavelength dependence (334-549 nm irradiation) of IPD were studied by both the classical visual grading method and by spectrophotometric scoring using the RS system. The spectral changes that occur at long wavelengths with IPD mimic the natural absorption spectrum of melanin. Therefore, the IPD was scored in terms of the apparent change in melanin optical density, using the method Kollias and Baqer [Photochem. Photobiol. 43, 49-54 (1986)], based on reflectance in the 620-720 nm range. The nonlinearity of the visual grading method is demonstrated. The degree of IPD is first-order with respect to delivered dose and saturates after high doses. The maximum amount of IPD attained at saturation is greater for shorter wavelengths. Extrapolation of the reflectance data suggests the longest wavelength capable of eliciting IPD is about 470 nm.

Photodynamic therapy with photofrin II induces programmed cell death in carcinoma cell lines.

The mode of cell death following photodynamic therapy was investigated from the perspective of programmed cell death or apoptosis. Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a) and rat mammary carcinoma (MTF7) were treated by photodynamic therapy. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder indicative of internucleosomal cleavage of DNA during apoptosis. The magnitude of the response and the photodynamic therapy dosage required to induce DNA fragmentation were different in PC3 and MTF7. The MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal response at the LD50 but had a marked response at the LD85. Thus, apoptosis did not ensue as quickly in PC3 as in MTF7. The H322a cells were killed by photodynamic therapy but failed to exhibit any apoptotic response. The results also suggested that apoptosis in these cell lines has a minor requirement for de novo protein synthesis and no requirement for de novo RNA synthesis. This study indicates that although apoptosis can occur during photodynamic therapy-induced cell death, this response is not universal for all cancer cell lines.

In vivo autofluorescence of an unpigmented melanoma in mice. Correlation of spectroscopic properties to microscopic structure.

Recently, fluorescence spectroscopy and imaging have been under investigation for in vivo diagnosis of several types of superficial cancer, including primary melanomas of the skin. Here we report on a detailed investigation of the autofluorescence properties of a K1735P melanoma implanted intradermally in the ears of syngeneic C3H/HeN mice. Although this tumour can produce melanin in some cases, it appeared as an unpigmented lesion in our experiments. Excitation-emission maps in the wavelength range of 360-700 nm were recorded from tumours and normal ears. The control ears and the tumour-bearing ears showed fluorescence in a broad range of excitation and emission wavelengths. Valleys of decreased fluorescence were observed in the 385-425 nm range and could be related to absorption of the excitation light by haemoglobin, oxyhaemoglobin and a third unknown absorber. The spectroscopic differences between the malignant melanoma and the control skin could be related to either differences in blood oxygenation or the tissue dimensions. However, no spectroscopic features were detected reflecting intrinsic differences between the melanoma and normal tissue.

Laser-tissue interactions. Photochemical, photothermal, and photomechanical.

An overview of laser-tissue interactions is presented in terms of the physical mechanisms of interaction, the time course of tissue response, and the level of biologic structure affected. The factors that affect dosimetry of photodynamic therapy are presented. Laser dosimetry for photothermal and photomechanical interactions is outlined.