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Biomedical subjects

K M Smith

Publications and source records attributed to K M Smith.

At least 19 recordsLinked to original sources
PubMed1992

Effectiveness of a lysyl chlorin p6/chlorin p6 mixture in photodynamic therapy of the subcutaneous 9L glioma in the rat.

A new photosensitizer, LCP, a combination of lysyl chlorin p6 and chlorin p6, was synthesized and tested for effectiveness in photodynamic therapy using s.c. implanted 9L glioma tumors in rats. Tumors were irradiated with 664-nm light 4 h after LCP injection. Mean intratumoral temperature elevations were less than 4 degrees C using a power density of 50 mW/cm2 for 33.3 min (100 J/cm2). Subsequent experiments examining histological changes and tumor regrowth used a power density of 50 mW/cm2 and total energy densities of 25, 50, and 100 J/cm2. Microscopically, an energy density-dependent coagulation necrosis of tumor cells occurred in treated tumors. Long term inhibition of tumor growth was achieved only at an energy density of 100 J/cm2. Side effects of treatment were seen only in the irradiated area and consisted of coagulation necrosis of normal tissues in rats treated at 50 and 100 J/cm2, including severe skin necrosis. Exposure of rats to fluorescent room light did not cause any macroscopically detectable skin damage. Our data indicate that photodynamic destruction of s.c. 9L glioma tumors using LCP as a photosensitizer results in significant tumor growth inhibition and that further study of LCP is warranted.

Animals↗
PubMed1992

Effects of anti-transferrin receptor antibodies on the growth of neoplastic cells.

One approach to creating a state of iron deprivation in tumors is to expose them to monoclonal antibodies against the transferrin receptor (ATRAs). This paper reviews the recent history of studies with ATRAs. Both multivalent (IgM or IgA) and bivalent (IgG) ATRAs exhibit anti-tumor activity in vitro and in vivo, but IgG ATRAs appear to be most effective when used with an iron chelator such as deferoxamine or when used in pairs. Much more information is needed in order to understand: (1) how ATRAs work by themselves and in conjunction with chelators; (2) why ATRAs differ from one another in terms of their inhibitory potency; (3) whether ATRAs can be used successfully in conjunction with other anti-tumor agents, and (4) why tumors exhibit marked differences in their sensitivity to the effects of ATRAs. The toxicity of iron deprivation arising from ATRA treatment alone seems modest, but only further experimental work in vivo and in phase-1 clinical trials can determine whether the most recent observations can be converted into truly useful therapeutic tools.

Animals↗
PubMed1991

Role of iron in T cell activation: TH1 clones differ from TH2 clones in their sensitivity to inhibition of DNA synthesis caused by IgG Mabs against the transferrin receptor and the iron chelator deferoxamine.

TH1 and TH2 helper T cell clones have been studied with respect to their sensitivity to inhibition of DNA synthesis by an IgG anti-transferrin receptor antibody (ATRA), the iron chelator deferoxamine, and the combination of the two reagents. TH1 clones are very sensitive to ATRA-mediated inhibition of DNA synthesis while TH2 clones are very resistant, but both TH1 and TH2 clones show significant down-modulation of surface transferrin receptors after ATRA exposure. TH2 clones exhibit larger chelatable iron storage pools than TH1 clones, however, and even partial chelation of TH2 cell storage iron does not fully convert a TH2 clone to the ATRA sensitivity pattern of a TH1 clone. It is therefore proposed that the greater resistance of TH2 clones to ATRA mediated inhibition derives from the combined effects of larger and less labile iron storage pools. These studies provide novel evidence indicating that nonuniform iron metabolism can exist within the T cell compartment and thus raise questions as to why such differences exist and how they can be integrated into models of the T cell activation process. These studies also suggest that the cell-mediated immune response in vivo, which is known to be sensitive to iron deficiency, may be evoked by effector cells which resemble TH1 clones insofar as iron metabolism is concerned.

Animals↗
PubMed1991

1H NMR study of the role of heme carboxylate side chains in modulating heme pocket structure and the mechanism of reconstitution of cytochrome b5.

1H nuclear magnetic resonance spectroscopy was used to assign the hyperfine-shifted resonances and determine the position of a side chain in the heme cavity of wild-type rat apocytochrome b5 reconstituted with a series of synthetic hemins possessing systematically perturbed carboxylate side chains. The hemins included protohemin derivatives with individually removed or pairwise shortened and lengthened carboxylate side chains, as well as (propionate)n(methyl)8-nporphine-iron(III) isomers with n = 1-3 designed to force occupation of nonnative propionate sites. The resonance assignments were effected on the basis of available empirical heme contact shift correlations and steady-state nuclear Overhauser effect measurements in the low-spin oxidized proteins. The failure to detect holoproteins with certain hemins dictates that the stable holoproteins, unlike the case of myoglobin, demand the axial iron-His bonds and cannot accommodate carboxylate side chains at interior positions in the binding pocket. Hence, the heme pocket interior in cytochrome b5 is judged much less polar and less sterically accommodating than that of myoglobin. The propionate occupational preference was greatest as the native 7-propionate site, but also possible at the nonnative crystallographic 5-methyl or 8-methyl positions. Only for a propionate at the crystallographic 8-methyl position was a significant perturbation of the native molecular/electronic structure observed, and this was attributed to an alternative propionate-protein hydrogen bond at the crystallographic 8-methyl position. The structures of the transient protein complexes detected only shortly after reconstitution reveal that the initial encounter complexes during assembly of holoprotein from apoprotein and hemin involve one of the two alternate propionate-protein links at either the 7-propionate or native 8-methyl position. In a monopropionate hemin, this leads to the characterization of a new type of heme orientational disorder involving rotation about a N-Fe-N axis.

Animals↗
PubMed1991

Effects of chronic and subchronic nicotine on tyrosine hydroxylase activity in noradrenergic and dopaminergic neurones in the rat brain.

Chronic nicotine (0.8 mg/kg by daily subcutaneous injection) over a 7 to 28-day period was found to increase the activity of tyrosine hydroxylase in predominantly noradrenergically innervated regions but not in dopaminergic projection areas. Increases in tyrosine hydroxylase activity were observed in dopaminergic cell body regions only after nicotine treatment for 3 to 5 days. The increase in tyrosine hydroxylase activity in noradrenergic neurones was evident first in the cell bodies in the locus coeruleus from 3 to 7 days, reaching 223% of control activities, and was followed by increases of up to 205% in the terminals up to 3 weeks later. It was then established that nicotine for 7 days was sufficient to increase the activity of the enzyme to the same extent in the terminals at 21 days even without further nicotine administration. This is consistent with axonal transport preceded by induction of the enzyme in noradrenergic cell bodies, whereas "delayed activation" might account for the transient effect seen in dopaminergic cell body regions. The response in the locus coeruleus to nicotine for 7 days was completely blocked by daily preinjection with mecamylamine but not with hexamethonium, which is consistent with the effect of nicotine on tyrosine hydroxylase being mediated by central nicotinic receptors.

Animals↗
PubMed1991

Regioselective photoreduction of zinc(II) porphyrins to give chlorins.

The ascorbic acid/organic base photoreduction of zinc(II) porphyrins was investigated. It was established that certain substituents can direct the photoreduction to the site of the macrocycle to which they are attached. For example, zinc(II) vinylporphyrins (8, 12, 16, 20) are photoreduced with cis stereochemistry on the ring bearing the vinyl group to give the corresponding chlorins. Zinc(II) acetylporphyrins (22, 24) were likewise reduced to chlorins such that cis-hydrogenation took place on the ring bearing the acetyl group. Zinc(II) formylporphyrins 33 also appear to reduce at the ring bearing the formyl group. When the zinc(II) acrylic porphyrin 28 was photoreduced, reduction did take place at the ring bearing the acrylic side chain, but migration of the acrylate double bond was very rapid, and the product isolated was the corresponding porphyrin propionate 30. Reduction of a zinc(II) porphyrin 35 bearing both a vinyl group and a nuclear carboxylic ester took place at the ring bearing the carboxylic ester. The reaction provides a general method for regioselective synthesis of chlorins from zinc(II) porphyrins without any evidence of formation of over-reduction products characteristic of many other procedures for formation of chlorins from porphyrin precursors.

Metalloporphyrins↗
PubMed1991

Chlorin and porphyrin derivatives as potential photosensitizers in photodynamic therapy.

In order to find a photosensitizer with better optical properties and pharmacokinetics than Photofrin II, a series of new photosensitizers related to methyl pheophorbide-a and chlorin-e6 were synthesized. These compounds absorb at substantially longer wavelengths (lambda max 660 nm) than does Photofrin II (630 nm) and show promise for use in photodynamic therapy. Among the porphyrins, we observed that long carbon chain ether derivatives are better photosensitizers than their ester analogs. These sensitizers were tested for in vivo photosensitizing activity vis-a-vis Photofrin II, using the standard screening system of DBA/2 mice bearing transplanted SMT/F tumors. Most of these photosensitizers were found to have better tumoricidal photosensitizing activity than Photofrin II and demonstrated more rapid attenuation of normal tissue photosensitivity with time after administration vis-a-vis Photofrin II.

Molecular Structure↗
PubMed1991

Dehydroandrographolide succinic acid monoester as an inhibitor against the human immunodeficiency virus.

Dehydroandrographolide succinic acid monoester (DASM) is the dehydroandrographolyl ester of succinic acid; and andrographolide, from which DASM is made, is the major diterpenoid lactone found in the Chinese medicinal herb, Andrographis paniculata. DASM has been found to be an inhibitor against the human immunodeficiency virus (HIV) in vitro. It was nontoxic to the H9 cell at the concentrations of 50-200 (average, 108) micrograms/ml and was inhibitory to the HIV-1 (IIIB) at the minimal concentration of 1.6-3.1 (average 2.0) micrograms/ml. It was also inhibitory to two other strains of HIV-1 and a strain of HIV-2. This inhibitory effect could also be demonstrated in cultures of activated human blood mononuclear cells; the 50% toxic dose and the 50% HIV inhibitory dose were about 200-greater than or equal to 400 and 0.8-2 micrograms/ml, respectively. At the subtoxic concentration, DASM partially interfered with HIV-induced cell fusion and with the binding of HIV to the H9 cell. Presumably, it also interfered with HIV replication at another unidentified step(s).

Antiviral Agents↗
PubMed1990

1H-NMR study of the mechanism of assembly and equilibrium heme orientation of sperm whale myoglobin reconstituted with protohemin type-isomers.

The products of the incorporation of various protohemin type-isomers into the heme pocket of sperm whale myoglobin were investigated by 1H-NMR in the met-cyano complexes, both immediately after reconstitution as well as at equilibrium. The type-isomers studied include those involving all possible interchanges of the two substituents on a given pyrrole. The protohemin-III and -XIII isomers, with true 2-fold symmetry, yielded only homogeneous products. Protohemins-XI, -XIV both exhibited two species after reconstitution, with one disappearing with time. Protohemin-I was the only asymmetric hemin that failed to exhibit two isomers initially. The orientation of the hemin within the pocket was established by nuclear Overhauser detected dipolar connectivities among heme substituents and between heme substituents and assigned heme pocket residues. At equilibrium, the heme orientations were dominated by the asymmetric propionate rather than vinyl dispositions on the hemin, with a clear preference for placing a propionate at the 8- vs. 5-methyl position of native myoglobin. For protohemin-XI, the propionates were found in the unexpected positions of the 7-propionate and 2-vinyl groups of native myoglobin, indicating that propionates can occupy positions well within the hydrophobic interior. The alternate heme orientation for the metastable intermediates detected for protohemin-XI and -XIV involved rotational isomerism about the alpha,gamma-meso axes bisecting the vinyl positions, but these two axes are at right angles to each other in the protein matrix. The fact that protohemin-XIV, but not protohemin-I, exhibits a reversed orientation as a reconstitution intermediate provides direct evidence that vinyl contacts, as well as propionate links, modulate the relative stabilities of the initial encounter complexes between hemin and apomyoglobin. The heme cavity molecular/electronic structure was found largely unperturbed for the complexes of the various protohemin type-isomers.

Animals↗
PubMed1990

1H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5.

The proton nuclear magnetic resonance spectra of the soluble fragment of native bovine and genetically engineered wild-type rat ferricytochrome b5 reconstituted with a wide variety of hemes chemically modified at 2- and/or 4-positions have been recorded and analyzed. While all but one nonsymmetric heme yielded comparable amounts of the two heme orientations immediately after reconstitution, the relative proportion of the two orientations at equilibrium varied widely. The unpaired spin density distribution in the heme pi system leads to substituent hyperfine shift patterns in these paramagnetic complexes that are completely diagnostic of the heme orientation in the protein matrix. An empirical assignment strategy is outlined and applied which allows unequivocal assignment of the absolute orientation of a derivatized heme within the protein matrix. Using a series of hemes lacking 2-fold symmetry solely due to a single substitution, the preferences for localized site occupation of vinyls, methyls, and hydrogens are developed. The large differences in relative stability of the two orientations of native protohemin in the two cytochromes b5 is shown to result from the additivity of localized effects for the bovine protein and the near cancellation of competing effects in the rat protein. The major determinant of the heme orientation is judged to be a repulsive interaction between a vinyl and a hydrophobic cluster of amino acids including positions 23 and 25. The differences in this heme orientational preference among bovine, rat, and chicken ferricytochromes b5 could be correlated with the relative steric bulk of the residues at positions 23 and 25.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Synergistic inhibition of lymphoid tumor growth in vitro by combined treatment with the iron chelator deferoxamine and an immunoglobulin G monoclonal antibody against the transferrin receptor.

Data are presented indicating that the growth of 5 out of 5 murine lymphoid tumors can be inhibited in a synergistic fashion in vitro by combined treatment with the iron chelator deferoxamine (DFO) and an immunoglobulin G (IgG) monoclonal anti-transferrin receptor antibody (ATRA). A two-way dose/response analysis shows that the ATRA becomes more efficient as an inhibitor with increasing doses of DFO. Flow cytometric studies further support the view that IgG ATRAS impair transferrin receptor (TR) function by causing TR down-modulation and degradation, even when the presence of DFO acts to promote increased cell surface TR expression. It is also shown that an IgG ATRA is nearly as effective as an IgM ATRA in inhibiting tumor cell growth when used in combination with DFO. Finally, studies with the iron chelator picolinic acid show that it produces only additive, or very slightly supra-additive, effects when used in combination with the ATRA. Therefore, these studies not only continue to suggest that combination chelator/ATRA therapy warrants further investigation as a tool in the therapy of hematopoietic malignancies, but also make the following new points: (1) the clinically familiar iron chelator deferoxamine, but not all iron chelators, produces synergistic inhibition of tumor growth in vitro with ATRAS; and (2) IgG ATRAS, which may be clinically more attractive reagents than IgA or IgM ATRAS because of better access to extra vascular tissue spaces, have unexpectedly been found to function as powerful growth inhibitors when used in combination with DFO.

Animals↗
PubMed1990

Horse heart myoglobin reconstituted with a symmetrical heme. A circular dichroism study.

Proton NMR studies on myoglobins and hemoglobins reconstituted with non-natural hemes, possessing different side chains in the pyrrolic rings, have provided interesting information for the understanding of the mechanism governing heme reorientation in the globin pocket, during synthesis of the native protein in vivo or in the reconstitution process in vitro. More recently, circular dichroism (CD) studies have been reported as a qualitative, alternative tool, with respect to 1H-NMR for detecting heme disorder in a reconstituted myoglobin or hemoglobin. In this paper, a CD study is reported on the reconstitution of horse heart myoglobin with protoheme XIII, a heme possessing true rotational symmetry about its alpha, gamma-meso axis. The results obtained show that the reconstitution product with this heme, which binds to the apoprotein with high affinity, not dissimilar from that of the natural heme, is characterized by a CD spectrum with bands possessing rotational strengths much lower than in the native protein. Furthermore, the CD changes detected as a function of time, during heme reorientation, in the case of natural heme, are absent when the apoprotein is reconstituted with protoheme XIII. These data provide independent evidence for reorientation of the natural heme, which follows its insertion into the protein matrix.

Animals↗
PubMed1990

Red shift of absorption maxima in chlorobiineae through enzymic methylation of their antenna bacteriochlorophylls.

The bacteriochlorophyll d producing photosynthetic green sulfur bacteria Chlorobium vibrioforme forma thiosulfatophilum strain NCIB 8327 and C. vibrioforme strain B1-20 respond to reduced light conditions in culture by performing methylations at the 4- and 5-substituents, for example, converting the 4-Et into 4-n-Pr, 4-i-Bu, and even 4-neoPn. During this process, the absorption maximum in living cells of C. vibrioforme strain B1-20 red shifts from 714 to about 728 nm. Eventually, the C. vibrioforme forma thiosulfatophilum strain NCIB 8327 culture carries out a delta-methylation to produce the bacteriochlorophylls c (lambda max ca. 750 nm); the new UC Davis bacteriochlorophyll c culture is named C. vibrioforme forma thiosulfatophilum strain D. It is possible that the homologation process increases hydrophobic interactions between individual BChl molecules, giving rise to larger aggregates in the antenna system. Alternatively, the additional methyl units attached to the 4-position shift the absolute configuration of the 2-(1-hydroxyethyl) group from pure R in the case of 4-Et to pure S in the case of 4-neoPn, which in turn might determine the size of the in vivo aggregates due to the intrinsic nature of the pigment protein system. It is suggested that the bacteriochlorophylls c from Chloroflexus aurantiacus strain J-10-fl and the bacteriochlorophylls e from Chlorobium phaeovibrioides might have undergone similar meso methylation as a response to external environmental pressure such as low light intensity.

Bacteria↗
PubMed1990

Biosynthetic studies of substituent homologation in bacteriochlorophylls c and d.

Administration of carbon-13 and carbon-14 labeled glutamate, glycine, and methionine to Chlorobium vibrioforme forma thiosulfatophilum strain D have demonstrated operation of the C5 and C1 metabolic pathways in bacteriochlorophyll c and bacteriochlorophyll d biosynthesis in this organism, with glutamate providing the delta-aminolevulinic acid for macrocycle synthesis and glycine providing the source of the extra homologation at the 4-, 5-, and delta-positions (via S-adenosylmethionine). Further evidence showing that the bacteria appear to adjust the homologue composition of their antenna bacteriochlorophylls in response to varying growth conditions is presented. Timing of these changes within a single culture is consistent with a light adaptation mechanism, which predicts that degree of alkylation is directly proportional to light intensity in the culture; other factors influencing pigment composition during the lifespan of a single culture may also be operating, and these are discussed.

Bacteria↗
PubMed1990

Porphyrin dimers as photosensitizers in photodynamic therapy.

Porphyrin dimers 9 with either linkages and possible isomers bis[1-[6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2- vinylporphin-4-yl]ethyl] ether (10) bis[1-[6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4- vinylporphin-2-yl]ethyl] ether (11), and 1-[6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-2-vinylporph in- 4-yl]ethyl 1-[6,7-bis[2-(methoxycarbonyl)ethyl]-1,3,5,8-tetramethyl-4-vinylporph in- 2-yl]ethyl ether (12) were synthesized from the corresponding (1-hydroxyethyl)vinyldeuteroporphyrin IX dimethyl esters (Hvd). The pure Hvd isomers 2-(1-hydroxyethyl)-4-vinyldeuteroporphyrin IX dimethyl ester (7) and 4-(1-hydroxyethyl)-2-vinyldeuteroporphyrin IX dimethyl ester (8) were obtained from 2-acetyl-4-(1-hydroxyethyl) deuteroporphyrin IX dimethyl ester (3) and 4-acetyl-2-(1-hydroxyethyl)deuteroporphyrin IX dimethyl ester (4). Porphyrins 3 and 4 were prepared either by partial reduction of 2,4-diacetyldeuteroporphyrin IX dimethyl ester (2) or by oxidation of hematoporphyrin IX dimethyl ester (1) by using tetra-n-propylammonium perruthenate (Prn4N)(RuO4) with N-methylmorpholine N-oxide as an oxidizing agent. The in vivo photosensitizing ability and therapeutic ratios of dimers 9-12 were compared with that of Photofrin II in the SMT-F tumor growing subcutaneously in DBA/2 Ha mice. These dimers were found to have better tumoricidal activity than Photofrin II with reduced skin phototoxicity.

Animals↗
PubMed1990

K+ (Rb+) transport by a mammary secretory cell apical membrane fraction isolated from goats' milk.

The transport of K+ (Rb+) by cell fragments isolated from goats' milk, which are believed to be pieces of mammary epithelial cell cytoplasm encapsulated by apical membrane, has been studied. K+ (Rb+) accumulation was time dependent and was virtually abolished by 0.1% Triton X-100 which indicates that K+ (Rb+) uptake is into a membrane-limited space rather than by binding. Replacing medium Cl- with SCN- increased whereas gluconate replacement of Cl- decreased K+ (Rb+) influx suggesting that uptake is sensitive to changes in membrane potential. Na+, K+, Li+ and Rb+ ions added to the incubation medium reduced the rate of K+ (Rb+) accumulation. K+ (Rb+) uptake was markedly reduced by both barium ions and quinine; the inhibitory effects were not additive suggesting they are acting at a single locus. Increasing the pH of the medium stimulated the Ba2(+)-sensitive component of K+ (Rb+) influx. The results show that the transport properties of the goats' milk membrane fraction used here are similar to the apical aspect of the intact mammary epithelium. Therefore this membrane preparation may be a good source and model to study mammary secretory cell apical membrane ion transport.

Animals↗
PubMed1990

Cellular localization of apolipoprotein D and lecithin:cholesterol acyltransferase mRNA in rhesus monkey tissues by in situ hybridization.

Apolipoprotein D (apoD) and lecithin:cholesterol acyl transferase (LCAT) are found on high density lipoprotein particles (HDLs) and have been postulated to form part of a complex involved in the transport of cholesterol from peripheral tissues to the liver for excretion. We have examined the sites of synthesis of the mRNAs for these two proteins in the rhesus monkey by in situ hybridization. ApoD mRNA-containing cells were widely distributed throughout peripheral tissues in interstitial and connective tissue fibroblasts often associated with blood vessels or capillaries. ApoD mRNA was also found localized in cells associated with peripheral nerves, neuroglial cells, cells in the subarachnoid space on the surface of the brain including the pial cells, perivascular cells, and scattered neurons in the brain. LCAT demonstrated a much more restricted pattern of synthesis and was found to be synthesized by hepatocytes, the basal cell layer of the epidermis, and in brain cell populations distinct from those that synthesize apoD. In the brain LCAT was synthesized by scattered neurons, neuroglial cells, ependymal cells, as well as a discrete cell layer in the cerebellum. ApoD has been shown to possess extensive homology to retinol binding protein, which has a binding pocket for vitamin A. We propose that apoD may also function to bind cholesterol or its derivatives in compartments not in direct contact with the blood. The findings of both apoD and LCAT synthesis in the brain suggest that they play a significant role in lipid transport in the brain.

Animals↗