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Biomedical subjects

K McMillan

Publications and source records attributed to K McMillan.

At least 37 records · Page 2Linked to original sources
PubMed1996

Binding of intermediate, product, and substrate analogs to neuronal nitric oxide synthase: ferriheme is sensitive to ligand-specific effects in the L-arginine binding site.

The electron paramagnetic resonance spectra of purified neuronal nitric oxide synthase indicates that the binding of ligands to the arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Four categories of high-spin complex can be distinguished; all are five-coordinate, and all retain the axial thiolate ligand, but they differ in their ligation geometries. These spectroscopic species reveal distinct local conformations which can be stabilized individually by the binding of L-arginine, N omega-hydroxy-L-arginine, N omega-methyl-L-arginine, and N omega-nitro-L-arginine. Other arginine analog inhibitors stabilize one or more of these states, revealing patterns based on the nature of substituents at the terminal amino group.

Animals↗
PubMed1996

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Animals↗
PubMed1996

Early diagnosis of upper aerodigestive tract cancer by autofluorescence.

OBJECTIVE: To explore the potential of autofluorescence spectroscopy as a tool for early detection of upper aerodigestive tract cancer. DESIGN: Autofluorescence spectral characteristics of 19 untreated oral and oropharyngeal lesions in 13 patients were studied with excitation wavelengths of 370 and 410 nm generated by a nitrogen pumped dye laser. Ten healthy volunteers were recruited to characterize the fluorescence spectra of normal mucosa at different oral sites and to study individual variations. Fluorescence intensity and line shape of the spectra from lesions were compared with the same parameters from the contralateral control site in the same individual. SETTING: Otolaryngology Research Center, Department of Otolaryngology-Head and Neck Surgery, New England Medical Center, Boston, Mass. RESULTS: The ratio of peak fluorescence intensities of the neoplastic lesions to contralateral normal control mucosa were consistently different compared with these ratios in benign lesions or normal mucosa. These differences were seen in 2 distinct regions of the fluorescence spectrum with both of the excitation wavelengths, but were more obvious with the excitation wavelength of 410 nm. Using these differences, we were able to correctly diagnose 17 of the 19 lesions studied, with 2 false-positive results. CONCLUSIONS: Neoplastic oral mucosa shows consistent differences in autofluorescence spectral intensity and line shape when compared with the normal mucosa in the same individual. These early results show that fluorescence spectroscopy may represent a useful technique for noninvasive early diagnosis of cancer of the upper aerodigestive tract.

Adult↗
PubMed1996

Characterization of tissue ablation with a continuous wave holmium laser.

BACKGROUND AND OBJECTIVE: The pulsed holmium laser is a promising tool for tissue ablation but possesses some limitations. For example, it is capable of producing significant mechanical damage in certain tissues in the form of fissures and fractures. Because longer pulse durations should reduce mechanical damage, this study examined the tissue effects produced by a prototype continuous wave holmium laser. STUDY DESIGN/MATERIALS AND METHODS: A prototype liquid nitrogen cooled holmium operating at 2.12 microns was used. The heat of ablation and ablation threshold were determined using a mass loss technique. Fresh pig skin was irradiated in air or under saline, and prepared for histologic analysis. Hemostasis was qualitatively assessed in vivo during incisions made in the skin, liver, and small intestine. RESULTS: Threshold radiant exposure and heat of ablation were calculated from the mass loss measurements to be 191 J/cm2 with a 95% confidence interval of -80-440 J/cm2. Residual thermal damage in skin ranged from 390 to 690 microns in saline and from 390 to 490 microns in air. Excellent hemostasis was achieved in all incisions. CONCLUSION: Using appropriate irradiation parameters, the continuous wave holmium laser produces tissue effects suitable for a general use surgical instrument. In addition, the laser source could become compact and inexpensive when diode-pumped and direct diode devices become available.

Air↗
PubMed1996

Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structure studies of a cysteine thiolate-liganded heme protein that hydroxylates L-arginine to produce NO. as a cellular signal.

The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca(2+)-calmodulin and/or tetrahydrobiopterin (BH4).

Animals↗
PubMed1995

Inhibition of nitric oxide synthase activity by Zn2+ ion.

We have found neural nitric oxide synthase (nNOS) activity to be completely and reversibly inhibited by Zn2+ ion with an apparent Ki of 30 microM. Zn2+ blocks NADPH-dependent reduction of heme iron in nNOS and also blocks the calmodulin-dependent superoxide-mediated cytochrome c reductase activity exhibited by nNOS. However, Zn2+ ion has no apparent effect on the calmodulin-independent direct reduction of cytochrome c by nNOS. Zn2+ ion induces perturbation difference spectra in nNOS characterized by the appearance of a peak at approximately 430 nm and a trough at approximately 395 nm, with an apparent spectral binding constant of 50 microM. These spectral changes are consistent with a Zn(2+)-dependent change in the spin-state equilibrium of the heme iron in nNOS. The spectral binding constant for L-arginine binding to nNOS (approximately 1.5 microM) is not significantly affected by the presence of 50 microM Zn2+, indicating that Zn(2+)-dependent inhibition of nNOS activity is not due to interference with substrate binding. The estimated maximal change in nNOS absorbance at approximately 418 nm caused by the L-arginine-dependent conversion of the ferric heme iron from hexacoordinate low-spin to pentacoordinate high-spin is increased by 50% in the presence of 50 microM Zn2+, which reflects the increased initial amount of low-spin ferric heme iron present. These data indicate that Zn(2+)-dependent inhibition of nNOS activity is due to binding of Zn2+ to the hemoprotein domain in the enzyme and that inhibition is associated with perturbations in the environment of the heme iron that appear to block its ability to mediate oxygen reduction.

Animals↗
PubMed1995

Characterization by electron paramagnetic resonance of the interactions of L-arginine and L-thiocitrulline with the heme cofactor region of nitric oxide synthase.

Nitric oxide synthase (NOS) catalyzes sequential NADPH- and O2-dependent mono-oxygenase reactions converting L-arginine to N omega-hydroxy-L-arginine and N omega-hydroxy-L-arginine to citrulline and nitric oxide. The homodimeric enzyme contains one heme/monomer, and that cofactor is thought to mediate both partial reactions. Here we show by electron paramagnetic resonance spectroscopy that binding of substrate L-arginine to neuronal NOS perturbs the heme cofactor binding pocket without directly interacting as a sixth axial heme ligand; heme iron is exclusively high spin. In contrast, binding of L-thiocitrulline, a NOS inhibitor, produces both high and low spin iron spectra; L-thiocitrulline sulfur is a sixth axial heme ligand in one, but not all, of the low spin forms. The high spin forms of the L-thiocitrulline NOS complex display a distortion in the opposite direction to that caused by L-arginine binding. The findings elucidate the binding interactions of L-arginine and L-thiocitrulline to neuronal NOS and demonstrate that each causes a unique perturbation to the heme cofactor pocket of NOS.

Animals↗
PubMed1995

Long-range effects on the retinal chromophore of bacteriorhodopsin caused by surface carboxyl group modification.

Carboxyl groups of bacteriorhodopsin (bR) that are modified by 1-ethyl-3-[3-(trimethylamino)-propyl]carbodiimide (ETC) have been identified. Reaction of deionized purple membrane with a 400-fold molar excess of ETC or [14C]ETC for 1 h at 0 degree C incorporates about 3.5 mol of ETC/mol of bR. Proteinase K cleavage of ETC-modified bacterioopsin (bO) produced small 14C-labeled peptides. Amino acid sequence analysis showed three major ETC-modified residues: Glu 234, Asp 38, and Glu 74. Proteolysis of purple membrane with papain removes the ETC site at Glu 234. Treatment of ETC-modified, papain-cleaved purple membrane with hydroxylamine removes half of the remaining ETC label. Subsequent cleavage with chymotrypsin, followed by amino acid sequence analysis, revealed that most of the remaining label was at Glu 74. bR modified by ETC primarily at Glu 74 displays two alterations in the retinal chromophore, located in the membrane interior at a distance more than 2 nm away from the modified carboxyl group. (1) The acid-induced purple-to-blue transition undergoes a shift in apparent pK from 3.2 to 2.3. (2) The second-order rate constant for chromophore regeneration from bO and retinal is diminished from 3600 to 1700 M-1 s-1 in membrane sheets. Most of the shift in the pK of the purple-to-blue transition can be explained by the quaternary ammonium ion of ETC attached to Glu 74 overlapping the postulated location of the guanidinium group of Arg 82.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Modular structure of neuronal nitric oxide synthase: localization of the arginine binding site and modulation by pterin.

A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds N omega-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH4. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH4 binding site by analogy to its homology with DHFR, but that tight binding of BH4 requires amino acids 220-577.

Amino Acid Oxidoreductases↗
PubMed1995

S-alkyl-L-thiocitrullines. Potent stereoselective inhibitors of nitric oxide synthase with strong pressor activity in vivo.

Nitric oxide synthase catalyzes the oxidation of a guanidino nitrogen of L-arginine to nitric oxide with concomitant formation of citrulline. Enzyme activity is inhibited by a variety of N omega-monosubstituted L-arginine analogs including N omega-alkyl-, N omega-amino-, and N omega-nitro-L-arginine derivatives. We report here that both constitutive and inducible isoforms of nitric oxide synthase are strongly inhibited by S-alkyl-L-thiocitrullines (N delta-(S-alkyl)isothioureido-L-ornithines) with n-alkyl groups of one to three carbons. These compounds represent a novel class of inhibitors and are the most potent nitric oxide synthase-inhibiting amino acids described to date. Inhibition is reversible, stereoselective, and competitive with L-arginine. Spectral studies show no direct interaction of inhibitor sulfur with heme iron, a result in contrast to that seen previously with the parent compound, L-thiocitrulline. The S-alkyl-L-thiocitrullines have strong pressor activity in normotensive control rats; S-methyl-L-thiocitrulline reverses hypotension in a rat model of septic peritonitis and in dogs administered endotoxin. These latter findings suggest that the inhibitors may have therapeutic utility in treating hypotension due to the overproduction of nitric oxide.

Amino Acid Oxidoreductases↗
PubMed1995

Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415.

The heme- and flavin-binding domains of constitutive rat neuronal nitric oxide synthase (NOS) were expressed in Escherichia coli as distinct polypeptides with properties characteristic of the intact enzyme. The amino-terminal heme-binding domain (residues 1-714) was expressed using the expression vector pCW. The denatured molecular mass of the expressed protein was 80 kDa, and the protein was shown to be immunoreactive to rabbit anti-NOS IgG. The NOS hemoprotein exhibited a ferrous-carbon monoxide difference spectrum with a wavelength maximum at 445 nm. Spectral perturbation with L-arginine and BH4 elicited a type I difference spectrum, confirming the presence of binding sites for these molecules within the N-terminal NOS polypeptide. Site-directed mutagenesis was applied to the putative axial heme ligand, cysteine-415, generating the histidine mutant, which confirmed the identity of the proximal ligand. NOS flavoproteins, with (C1, residues 715-1429) and without (C2, residues 749-1429) an amino-terminal calmodulin-binding motif, were expressed using the vector pPROK-1. The C1 and C2 flavoproteins were immunoreactive to anti-NOS IgG and were sized at approximately 80 kDa. Both of the purified flavoproteins exhibited optical absorbance properties typical of a flavin prosthetic group, with wavelength maxima at 380 and 450 nm, and were competent in NADPH-dependent electron transfer to cytochrome c, with observed rates of approximately 2-4 mumol/min/mg. The bacterial expression of the NO synthase heme-binding oxygenase and flavoprotein oxidoreductase domains as isolated proteins with specific properties of the intact enzyme represents an important development in structure-function studies of this complex enzyme.

Amino Acid Oxidoreductases↗
PubMed1995

Pharmacokinetics, dosimetry and toxicity of holmium-166-DOTMP for bone marrow ablation in multiple myeloma.

UNLABELLED: In this Phase I clinical trial, six multiple myeloma patients who had not responded to conventional therapy and were scheduled for bone marrow transplantation received a bone-seeking radiopharmaceutical for bone marrow ablation. The pharmacokinetics, dosimetry, and toxicity of this radiopharmaceutical were studied. METHODS: Patients received from 519 mCi to 2.1 Ci (19.2 GBq to 77.7 GBq) of holmium-166 (166Ho) complexed with a bone-seeking agent, DOTMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylene-phosphonic acid). The reproducibility of pharmacokinetics from multiple injections of 166Ho-DOTMP administered to these myeloma patients was demonstrated from blood (r2 = 0.926) and whole-body retention (r2 = 0.983), which allowed therapeutic parameters to be determined from a diagnostic study. RESULTS: Over 50% of the 166Ho-DOTMP injected dose was excreted within 2-3 hr postinjection, increasing to 75%-85% over a 24-hr period. Rapid blood clearance minimized radiation dose to nontarget tissue: less than 10% of the injected activity was retained in the blood pool at 1 hr postinjection, and less than 2% remained after 5 hr. The total radiation absorbed dose delivered to the bone marrow for the six patients ranged from 7.9 Gy to 41.4 Gy. CONCLUSION: All patients demonstrated severe bone marrow toxicity with a white blood cell (WBC) count < 1,000 cells/microliters, two patients exhibited marrow ablation (WBC count < 100 cells/microliters), and no other toxicity > or = grade 2 was observed in any of the patients.

Bone Marrow↗
PubMed1994

L-thiocitrulline. A stereospecific, heme-binding inhibitor of nitric-oxide synthases.

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (NO). The enzyme is inhibited by a variety of N omega-monosubstituted L-arginine analogs, and some of these compounds are useful in reversing pathologies associated with the overproduction of NO (e.g. the hypotension of septic shock). We report here that L-thiocitrulline (gamma-thioureido-L-norvaline) is a potent, stereospecific inhibitor of the constitutive brain and endothelial isoforms of NOS as well as the isoform induced in vascular smooth muscle cells by lipopolysaccharide and interferon-gamma. Steady state kinetic studies show L-thiocitrulline inhibition is competitive with L-arginine (Ki approximately 4-20% of KArgm), indicating that initial binding is as a substrate/product analog. In contrast to L-arginine and N omega-methyl-L-arginine, the prototypic NOS inhibitor, L-thiocitrulline binding elicits a "Type II" difference spectrum, indicating a high spin to low spin transition of the iron in the heme cofactor. This finding suggests that L-thiocitrulline is contributing the sixth ligand to heme iron, probably through the thioureido sulfur. Such interaction with heme iron neither stimulates nor inhibits the direct flavin-mediated cytochrome c reduction activity of the enzyme, but it does inhibit heme-dependent superoxide formation. In vivo, L-thiocitrulline is a potent pressor agent in both normal and endotoxemic rats, the latter finding suggesting utility in treating the hypotension of septic shock.

Amino Acid Oxidoreductases↗
PubMed1994

Activation of myosin light chain kinase and nitric oxide synthase activities by calmodulin fragments.

We have investigated the abilities of calmodulin (CaM) tryptic fragments 1-75 (TRCI) or 78-148 (TRCII) to activate gizzard smooth muscle myosin light chain kinase (gMLCK), rabbit skeletal muscle myosin light chain kinase (skMLCK), and neural nitric oxide synthase (nNOS) activities. Our results indicate for all three enzymes that binding of CaM follows an ordered mechanism wherein the C-terminal lobe, represented by TRCII, binds specifically to a site we designated as A, followed by binding of the N-terminal lobe, represented by TRCI, to a site designated as B. With TRCII and TRCI bound to their respective sites, skMLCK and gMLCK activities are both activated to about 80% of their maximum levels. Occupancy of both sites in the MLCK enzymes by TRCI results in only low levels of enzyme activation; occupancy of both sites by TRCII also results in low levels of gMLCK activity, but activates skMLCK activity to 65% of the maximum level. With TRCI bound at site B and either TRCII or TRCI bound at site A, nNOS activity is 50% of the maximum level. Apparent dissociation constants for TRCII binding to site A and TRCI binding to site B are, respectively; 0.3 and 3 microM (skMLCK); 1.2 and 0.8 microM (gMLCK); 10 nM and 150 microM (nNOS). Our results demonstrate that the CaM lobes can make distinct contributions to binding and/or activation of different CaM-dependent enzymes and that the tethering function of the central helix can be mimicked by sufficiently high concentrations of the CaM fragments. We have modeled tethering as if it stabilizes the CaM-enzyme complex by creating a high effective concentration of the N-terminal lobe. Calculated values for this concentration term indicate essentially identical contributions by the central helix to the observed nanomolar dissociation constants of the three CaM-enzyme complexes examined.

Amino Acid Oxidoreductases↗