Biomedical subjects
Publications and source records attributed to D E Harris.
We report the characterization of cDNAs encoding two actin isoforms (alpha-smooth muscle and beta-non-muscle) from the rabbit uterus. These isoforms have extensive amino acid and nucleotide sequence homology to the corresponding mouse and human isoforms.
The length vs. active force relationship (L-F) may provide information about changes in smooth muscle contractile protein interactions as muscle length changes. To characterize the L-F in single toad stomach smooth muscle cells, cells were attached to a force measurement system, electrically stimulated, and isometric force and elastic modulus (an estimate of the number of attached cross bridges) determined at different cell lengths. Cells generated maximum stress (Pmax = 152.5 mN/mm2) and elastic modulus (Eact = 0.68 x 10(4) mN/mm2) at their rest length (Lcell = 78.0 microns; distance between cell attachments). At shorter lengths, active force and elastic modulus declined proportionally with active force eliminated at 0.4 Lcell. Stretching the relaxed cells up to 1.4 Lcell shifted the subsequent L-F along the length axis by the amount of the stretch but did not change Pmax or the shape of the L-F. In activated cells, force was a function of cell length rather than of shortening history. We interpret these findings as evidence that 1) Lcell is close to the optimum length for force generation, 2) the decline in force at lengths less than Lcell results from a reduced number of attached cross bridges, and 3) stretching relaxed smooth muscle cells may not move the contractile units to new positions on their L-F.
Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)
In single smooth muscle cells, shortening velocity slows continuously during the course of an isotonic (fixed force) contraction (Warshaw, D.M. 1987. J. Gen. Physiol. 89:771-789). To distinguish among several possible explanations for this slowing, single smooth muscle cells were isolated from the gastric muscularis of the toad (Bufo marinus) and attached to an ultrasensitive force transducer and a length displacement device. Cells were stimulated electrically and produced maximum stress of 144 mN/mm2. Cell force was then reduced to and maintained at preset fractions of maximum, and cell shortening was allowed to occur. Cell stiffness, a measure of relative numbers of attached crossbridges, was measured during isotonic shortening by imposing 50-Hz sinusoidal force oscillations. Continuous slowing of shortening velocity was observed during isotonic shortening at all force levels. This slowing was not related to the time after the onset of stimulation or due to reduced isometric force generating capacity. Stiffness did not change significantly over the course of an isotonic shortening response, suggesting that the observed slowing was not the result of reduced numbers of cycling crossbridges. Furthermore, isotonic shortening velocity was better described as a function of the extent of shortening than as a function of the time after the onset of the release. Therefore, we propose that slowing during isotonic shortening in single isolated smooth muscle cells is the result of an internal load that opposes shortening and increases as cell length decreases.
A single round of Edman degradation was employed to remove the NH2-terminal valine from isolated alpha chains of human hemoglobin. Reconstitution of normal beta chains with truncated or substituted alpha chains was used to form truncated (des-Val1-alpha 1) and substituted ([[1-13C]Gly1]alpha 1) tetrameric hemoglobin analogs. Structural homology of the analogs with untreated native hemoglobin was established by using several spectroscopic and physical methods. Functional studies indicate that the reconstituted tetrameric protein containing des-Val1-alpha chains has a higher affinity for oxygen, is less influenced by chloride ions or 2,3-bisphosphoglycerate, and shows lower cooperativity than native hemoglobin. These results confirm the key functional role of the alpha-chain NH2 terminus in mediating cooperative oxygen binding across the dimer interface. The NH2-terminal pK1/2 value was determined for the [13C]glycine-substituted analog to be 7.46 +/- 0.09 at 15 degrees C in the carbon monoxide-liganded form. This value, measured directly by 13C NMR, agrees with the determination made by the less-direct 13CO2 method and confirms the role of this residue as a contributor to the alkaline Bohr effect; however, it is inconsistent with the presence of an NH2-terminal salt bridge to the carboxylate of Arg-141 of the alpha chain in the liganded form.
We have prepared a semisynthetic analogue of fully acetimidylated horse cytochrome c, a complex in which the peptide bond between residues glycine-37 and arginine-38 is lacking. In contrast with the complex that we have previously described [Harris & Offord (1977) Biochem. J. 161, 12-25], in which the break in continuity is between residues arginine-38 and lysine-39, the new analogue has a nearly normal redox potential, and can more fully restore succinate oxidation to mitochondria depleted of cytochrome c. Studies of this and other analogues lead us to propose an explanation for the low biological activity of complex (1-38)-(39-104) and a role for the invariance of arginine-38.
The synthesis of a series of myoglobins substituted in the amino-terminal residue to provide variation in the aliphatic nature of the side chain and enrichment in 13C was accomplished by semisynthetic methods. The replacements for valine, the native first residue, included 13C-enriched glycine, alanine, valine, leucine, and isoleucine. The products were extensively characterized and found to be virtually indistinguishable by most physical methods. 13C NMR spectroscopy showed significant differences in the amino-terminal pK value, ranging from 7.72 for [Gly1]myoglobin to 7.15 for [Leu1]myoglobin. Consideration of the electrostatic effects of the charge matrix indicated a balance of interactions at this site not significantly altered by these variations in the side chain. By examination of the crystal structure, consideration of earlier work regarding the interactions of the side chain of Leu-2, and data regarding the motions of the terminal residue, it was concluded that the interaction of the side chain of the first residue with the hydrophobic cluster formed primarily by close contact of invariant residues Leu-2 and Leu-137 was the primary cause for the reduction in terminal pK values seen for the larger aliphatics. By restricting the freedom of the residue, this interaction limits the available hydration volume and consequently favors the unprotonated form of the amine. The concurrent observation of both functional elements in the series of alpha-amino-terminal residues brings out the interrelated consequences for the two categories of solvent interactions controlling structural and functional properties in a graded way.
The amino acid sequences of the myoglobins of two rodents, the casiragua and the house mouse, have been determined. The myoglobin of casiragua differs from that of viscacha (another hystricomorph) at 6 positions. Mouse myoglobin differs from that of mole-rat (another myomorph) at 17 positions, whereas casiragua and mouse differ at 22 positions. Mouse myoglobin possesses several features unique among all known myoglobins (Gly 31, Cys 66, Thr 74 and Glu 113) and one substitution unique among known mammalian myoglobins (Glu 53).
We have re-examined the acetimidylation and subsequent deprotection of cytochrome c by published methods in the light of recent findings on the tendency of protein acetimidylation reactions to yield side products of differing net charges. We find that the protection methods do indeed yield a mixture of products, some of which have considerably diminished biological activity. Our observations support a postulated mechanism for the generation of side products, and we have been able to identify the major factor responsible for their formation by published methods. The deprotection method appears to be free of side reactions. We describe a new procedure for acetimidylation that will produce fully N-epsilon-acetimidylated cytochrome c. This derivative, lacking detectable side products and having good biological activity, is useful for structure-function studies and as an intermediate in the semisynthesis of cytochrome c analogues.
1H-n.m.r. and 13C-n.m.r. spectroscopy of horse cytochrome c and 1H-n.m.r. spectroscopy of the lysine-modified proteins N epsilon-acetimidyl-, N epsilon-amidino-, N epsilon-trifluoroacetyl- and N epsilon-maleyl-cytochrome c have shown that, although the lysine modifications do not greatly perturb the protein structure at pH7 and 27 degrees C, at higher temperature or at alkaline pH some parts of the structure are markedly perturbed. At pH7 and 27 degrees C the region of the protein about Ile-57 is affected in all the modified proteins, though not all to the same degree. N epsilon-Maleylation most seriously affects the protein structure, and the fully maleylated protein is readily unfolded. At 27 degrees C all four of the tyrosine residues of native horse cytochrome c have pKa values above 11, but in N epsilon-acetimidyl-cytochrome c the pKa of one tyrosine residue is 10.2.
N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole epsilon-amino group. N epsilon-Acetimidoglucagon was isolated from the crude acetimidoglucagon mixture by anion-exchange chromatography at pH 9.4, producing a derivative which was identical with native glucagon on isoelectric focusing and which by amino acid analysis had greater than 98% of the lysine blocked. The yield was greater than that obtained when tetrahydrophthalic anhydride was used as a chromatographic handle to remove peptides with unreacted amino groups. N epsilon-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic N alpha-biotinyl-N epsilon-acetimidoglucagon, prepared by reacting (N-hydroxysuccinimido)biotin with N epsilon-acetimidoglucagon and purified by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2) and exhibited 1.2% of the binding affinity, 2.4% of the biological potency, and 30% of the maximum activity of the native hormone. Preliminary fluorescence microscopy demonstrated binding of N alpha-biotinyl-N epsilon-acetimidoglucagon to glucagon specific receptors following exposure to fluorescein-labeled avidin. Capping of labeled receptors could be visualized with time. (Des-His1)N epsilon-acetimidoglucagon, prepared via a manual Edman degradation of N epsilon-acetimidoglucagon and isolated by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2). The second residue, serine, has also been removed. Semisynthetic coupling of alternative residues to such derivatives will provide insight into the role of the amino-terminal residues in mediating the biological actions of the hormone.
The thiocyanate method for stepwise degradation of peptides from their COOH termini [Stark, G. R. (1968) Biochemistry 7, 1796] has been investigated. The method involves first the reaction of the COOH-terminal residue with thiocyanate in an activation solvent of acetic acid and acetic anhydride and then cleavage of the COOH-terminal residue as its 2-thiohydantoin by acetohydroxamate in aqueous solution. The two steps of the degradation have been studied by using model peptides, and conditions have been developed for the rapid efficient removal and identification of the COOH-terminal residue of short peptides. The methods have been applied to peptides that have been covalently attached to insoluble supports. In this solid phase version of the degradation, a highly substituted porous glass activated with N,N'-carbonyldiimidazole has been prepared for use as the insoluble support. A number of peptides have been coupled to the porous glass, and several rounds of the degradation have been performed on immobilized peptides. High-pressure liquid chromatography provides a rapid, sensitive identification method for the 2-thiohydantoins. In addition, gas-liquid chromatography of the amino acid 2-thiohydantoins and reconversion to the parent amino acid have been used to identify the cleaved residues. The method of sequential degradation has been applied to a number of short model peptides such as Gly-Leu-Tyr, Met-enkephalin, and Val-Leu-Ser-Glu-Gly and has been used to determine the COOH-terminal sequence of 4 residues of a 22-residue cyanogen bromide fragment of pygmy sperm whale myoglobin.
A unique experimental model is described, where natural immunologic tolerance to a well-defined soluble native antigen (murine C5) is examined in congenic strains of mice that differ only by the presence or the absence of C5. A highly sensitive hemolytic assay was developed to detect nanogram amounts of C5 as well as an assay of anti-C5 inhibition of C5 hemolytic activity. The latter was more sensitive than immunodiffusion. Two reciprocal approaches were used to study the cellular basis of tolerance in irradiated hosts of either strain. In the first, lymphoid cells from either strain were transferred to irradiated B10.D2OSN hosts that were lacking C5 and so would not hinder detection of anti-C5 antibody upon challenge with murine C5. Second, lymphoid cells from either strain were transferred to irradiated B10.D2NSN hosts, whose native C5 provided the antigenic stimulus. The immune response of whole nonadherent spleen cell suspension as well as mixtures of T and B cells (separated on the basis of surface immunoglobulin) from either strain were studied. In addition, the duration of tolerance and the antigen requirement to maintain it in irradiated C5-deficient hosts repopulated with C5-sufficient spleen cells was examined. The positive control of irradiated C5-deficient hosts repopulated with syngeneic spleen cells showed a primary and secondary response to immunization. In contrast, C5-sufficient spleen cells failed to respond both in the primary and the secondary response. Because the unresponsiveness was not caused by antigen carryover and was not antigen specific, it represents central tolerance. In C5-sufficient irradiated hosts (where immunization was not required and antigen was present in natural form and physiological concentration), transfer of C5-deficient cells mediated a drop in C5 levels to 10-20% of that noted in unreconstituted controls. T and B cell mixing experiments from the two strains into deficient or sufficient hosts demonstrated that tolerance is T cell dependent and that C5-sufficient or -deficient B cells could cooperate with nontolerant C5-sufficient T cells to produce significant anti-C5 antibody or mediate a significant drop in C5 levels. In addition, the presence of antigen was necessary to maintain tolerance. In conclusion, these results show that (a) natural tolerance to C5 is an active process that is T cell dependent and requires the presence of antigen; (b) in this natural model, clonal abortion does not seem to occur; and (c) both tolerant and nontolerant B cells retain the capacity to produce autoantibody.
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A retrospective cohort study was performed in Hawaii among 4,779 male shipyard workers exposed to asbestos and 2,757 similar male workers without known asbestos exposure. Observed deaths from cancer and other causes in the 2 groups were compared with expected deaths on the basis of the general population of Hawaii by the use of a modified life-table method of analysis. A risk ratio for lung cancer of 1.7 was found for the exposed group after 20 or more years of follow-up. No increased risk for lung cancer was seen in the nonexposed group. These findings could not be attributed to differences in smoking habits in the 2 shipyard groups or between the shipyard groups and the general population. Because the maximum duration of follow-up for this analysis was 24 years, greater risks for lung cancer may be seen in the exposed group when the follow-up period is extended.
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The gas chromatographic behavior of 32 carbamate pesticides was investigated using the Hall electrolytic conductivity detector. Relative retention indices were successfully determined for 24 carbamates on six different columns. Columns investigated included Ultra-Bond, 3% OV-101 on Ultra-Bond, 1% OV-17 on Ultra-Bond, 1% OV-210 on Ultra Bond, 1% Carbowax 20M on Ultra-Bond and 0.5% OV-210 + 0.65% OV-17 on Ultra-Bond. Chemical-ionization gas chromatography-mass spectrometry was used to verify that the carbamates were chromatographed intact. Chemical-ionization mass spectra are reported. Analytical procedures are demonstrated for the determination of carbamate residue in soil.
A specific portion of our environment has been contaminated with Kepone, or chlordecone. Additionally, some specific human exposures to high concentrations of Kepone have been confirmed. Gas chromatography mass spectrometry involving chemical ionization and high resolution mass spectrometry were used to detect, identify and confirm the presence of Kepone, Kepone photoproducts and a reduction product of Kepone in environmental and human samples. Field desorption, field ionization and electron impact mass spectrometric methods, as well as infrared and nuclear magnetic resonance techniques were used to characterize and identify Kepone hydrate and hemiketal in benzene and methanol solutions, respectively.