On following the God of technology.
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Anesthesiology↗
Biomedical subjects
L Callahan
Publications and source records attributed to L Callahan.
Dibasic amines as competitive ions improve the resolution between polyanionic nucleotides.
Aliphatic diamines when used as single ion pairing reagents were capable of resolving 3'-,5'- and 2'-,5'- nucleotidyl diphosphates from one another while conventional ion pairing reagents did not separate these positional isomers. The use of 1,2-diamines resulted in the greatest resolution while increasing spacing between the amino groups progressively reduced the resolution while increasing the retention volume. A competitive ion pairing system was also developed using triethylamine as an additional ion pairing reagent. Using this system ethylenediamine, 1,2- and 1,3-diaminopropane were nearly equivalent in their ability to resolve adenosine 3'-phosphate 5'-phosphate, from adenosine 2'-phosphate 5'-phosphate, and adenosine 3'-phosphate 5'-beta-methylenephosphosulfate (3'-mePAPS) from adenosine 2'-phosphate 5'-beta-methylenephosphosulfate (2'-mePAPS), respectively. The ability to easily resolve these positional isomers allows the use of a more simplified synthetic procedure that does not involve the use selective protecting groups to specifically phosphorylate the 2' or 3' hydroxyl group. We have used this procedure on a semipreparative scale to obtain small quantities of both mePAPS and 2'-mePAPS for use in enzymatic studies.
Adenine Nucleotides↗
Synthesis and utilization of a nonhydrolyzable phosphoadenosine phosphosulfate analog.
3'-Phosphoadenosine 5'-phosphosulfate (PAPS) functions as the high-energy sulfate donor for sulfate ester synthesis in all higher organisms. This activated sulfate, like its adenosine 5'-phosphosulfate precursor, is both chemically labile and vulnerable to sulfohydrolase degradation. These obstacles have limited the utility of the native PAPS in the purification and mechanistic description of the numerous PAPS-utilizing enzymes. This paper describes the synthesis of the 2'- and 3'-isomers of a nonhydrolysable, and thus stable, PAPS analog, beta-methylene-PAPS, from the previously described beta-methylene-APS (L. Callahan et al., Anal. Biochem. 177, 67-71, 1989). The method involves phosphorylation of beta-methylene-APS with trimetaphosphate and separation of the resulting mixed 2'(3')-isomers by ion-pair reverse-phase HPLC. The utilization of this analog as an inhibitor of APS kinase and PAPS translocase, two of the numerous PAPS-utilizing activities, as well as an affinity ligand for purification of APS kinase, is described.
Animals↗
Molecular structure of deoxycytidyl-3'-methylphosphonate (RP) 5'-deoxyguanidine, d[Cp(CH3)G]. A neutral dinucleotide with Watson-Crick base pairing and a right handed helical twist.
The crystal structure of d[Cp(CH3)G] has been determined as part of a project to study the mechanism of the B----Z transition in DNA. The asymmetric unit contains two dinucleotides and the equivalent of 7.5 water molecules, partially disordered over 12 definable positions. The two symmetry-independent dinucleotides form a duplex with Watson-Crick base-pairing and a right-handed helical sense. Comparison with previously determined structures of the B and A conformation showed that this duplex is closer to B than to A but significantly different from B. It corresponds to a stretched out helix with a 4 A rise per base pair and a helical twist of 32 degrees. This structure may serve as a model for the bending of DNA in certain situations. The configuration at the methyl phosphonate is RP, and a mechanism, based on this assignment, is presented for the B----Z transition in DNA.
Base Composition↗
Synthesis and properties of a nonhydrolyzable adenosine phosphosulfate analog.
Initial activation of inorganic sulfate for subsequent synthesis of sulfated biomolecules requires the action of ATP-sulfurylase to generate adenosine 5'-phosphosulfate (APS). This activated sulfate intermediate is both chemically labile and susceptible to enzymatic degradation. Consequently, it has not proven useful as a ligand for either purification or characterization of the various APS-utilizing enzymes. For these purposes, a stable analog of APS was required. This paper describes the simple and efficient synthesis and structural confirmation of a nonhydrolyzable APS analog, beta-methylene APS, with an overall molar yield of 40-50%. The method involves nucleophilic substitution of the chlorine moiety of a 5'-chloromethylphosphonate ester of 2',3'-O-isopropylidene adenosine by a sulfite ion. We also report the initial utilization of this compound as an inhibitor in kinetic trials of both ATP-sulfurylase and APS kinase and as an affinity ligand for the purification of these two APS-utilizing enzymes from cartilaginous tissue.
Adenosine Monophosphate↗
Rubella virus: mechanism of attenuation in the vaccine strain (HPV77).
The vaccine type (HPV77 strain) of rubella virus replicates slower and manifests a delayed appearance of cytopathic effect in Vero-76 cells as compared to wild-type virus (M33). The change in cytopathic effect coincides with the delayed appearance of both genomic and subgenomic RNA as well as viral structural proteins in the cell. The delay in the appearance of the viral proteins in the cells was also evident when the cells infected with the vaccine-type virus were treated with the lysosomotropic agent such as chloroquine. Binding studies using [35S]methionine-labeled virus showed that the vaccine-type virus bound to the cells poorly and the binding was not completely competed out with the cold virus.
Animals↗
Hemodynamic, renal, and endocrine effects of 4-h infusions of human atrial natriuretic peptide in normal volunteers.
A synthetic human atrial natriuretic peptide of 26 aminoacids [human (3-28)ANP or hANP] was infused into normal male volunteers. Six subjects were infused for 4 h at 1-wk intervals with either hANP at the rate of 0.5 or 1.0 microgram/min or its vehicle in a single-blind randomized order. Human (3-28)ANP at the dose of 0.5 microgram/min raised immunoreactive plasma ANP levels from 104 +/- 17 to 221 +/- 24 pg/ml (mean +/- SEM), but it induced no significant change in blood pressure, heart rate, effective renal plasma flow, glomerular filtration rate, or renal electrolyte excretion. At the rate of 1.0 microgram/min, human (3-28)ANP increased immunoreactive plasma ANP levels from 89 +/- 12 to 454 +/- 30 pg/ml. It reduced effective renal plasma flow from 523 +/- 40 to 453 +/- 38 ml/min (P less than 0.05 vs. vehicle), but left glomerular filtration rate unchanged. Natriuresis rose from 207 +/- 52 to 501 +/- 69 mumol/min (P less than 0.05 vs. vehicle) and urinary magnesium excretion from 3.6 +/- 0.5 to 5.6 +/- 0.5 mumol/min (P less than 0.01 vs. vehicle). The excretion rate of the other electrolytes, blood pressure, and heart rate were not significantly modified. At both doses, human (3-28)ANP tended to suppress the activity of the renin-angiotensin-aldosterone system. In 3 additional volunteers, the skin blood flow response to human (3-28)ANP, infused for 4 h at the rate of 1.0 microgram/min, was studied by means of a laser-doppler flowmeter. The skin blood flow rose during the first 2 h of peptide administration, then fell progressively to values below baseline. After the infusion was discontinued, it remained depressed for more than 2 h. Thus, in normal volunteers, human (3-28)ANP at the dose of 1.0 microgram/min produced results similar to those obtained previously with rat (3-28)ANP. It enhanced natriuresis without changing the glomerular filtration rate while effective renal plasma flow fell. It also induced a transient vasodilation of the skin vascular bed.
Adult↗
Competence models: from theory to practical application.
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Clinical Competence↗
Anatomy of axolotl flank integument during limb bud development with special reference to a transcutaneous current predicting limb formation.
We have compared the anatomy of immature axolotl integument from limb-forming regions with adjacent non-limb-forming regions of the flank, concentrating on the earliest stages of limb bud development. We have extended these observations to include prominent buds just prior to their differentiation. At the ultrastructural level, we note striking differences between these two regions of skin, including a complete loss of hemidesmosomes and tonofilaments in the basal cells of the epidermis; a marked deterioration of the basal lamella; and focal areas of desquamating cells in the apical regions of the bud-all characteristics of limb-forming regions. These observations were made in the same larvae which provided measurements of a steady endogenous electric (ionic) current that either was coincident with or predicted the area of limb bud outgrowth (Borgens et al.: J. Exp. Zool. 228:491-503, 1983). We discuss these physiological measurements, the changes in the anatomy of the bud-forming region, and the relevance of these observations to our theory of early limb formation.
Ambystoma↗
B- to Z-DNA transition probed by oligonucleotides containing methylphosphonates.
The simulation of the B--Z-DNA transition by using space-filling models of the dimer d(C-G) shows the possibility of hydrogen-bond formation between the N-2 amino group of the partially rotated guanine and one of the 5'-phosphate oxygens of deoxyguanylic acid. To probe the importance of this postulated interaction, analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, which replaced the first 5'-phosphate linkage of deoxyguanosine. The CD spectra in high salt concentration showed that the hexamer containing a methylphosphonate linkage with the RP stereochemistry formed Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage with the SP stereochemistry did not form Z-DNA. These results are consistent with a mechanism in which an interaction between the N-2 amino group of guanine and the prochiral SP oxygen of deoxyguanosine 5'-phosphate kinetically controls the formation of Z-DNA. A water bridge between the N-2 amino group of guanine and the 3'-phosphate oxygen of deoxyguanylic acid has been implicated in the stabilization of Z-DNA. To probe the importance of this water bridge, two additional analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, that replaced the first deoxyguanosine 3'-phosphate. The CD spectra showed that the hexamer containing a methylphosphonate linkage of the RP stereochemistry underwent the transition to Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage of the SP stereochemistry underwent the transition to Z-DNA to a 35% lesser extent. Thus the water bridge involving the prochiral SP oxygen provides modest stabilization energy for Z-DNA. These studies, therefore, suggest that the B--Z-DNA transition is regulated both thermodynamically and kinetically through hydrogen-bond interactions involving phosphate oxygens and the N-2 amino group of guanine.
Base Composition↗
Comparative sensitivity of different methods to detect and quantify circulating fibrinogen/fibrin split products.
This study compared the sensitivity of three methods: staphyloccocal clumping test, SCT (Sigma and Calbiochem-Behring, CBC, reagents); Thrombo-Wellcotest (TWT); and Dade fibrinogen degradation products detection set, to quantify fibrinogen/fibrin split products (FSP) in blood samples from 696 patients and 124 normal donors using fibrinogen as the reference value. The Dade method gave quantitative results closely approaching the stated amount of fibrinogen. The SCT using Sigma reagents gave higher "fibrinogen" values, while the CBC reagents gave markedly lower "fibrinogen" values. The TWT detected only 25% of the fibrinogen standard. Detection of FSP following plasmin digestion of fibrinogen varied considerably for each test. The TWT, insensitive to most of the native fibrinogen, detected most of the FSP following only 15 minutes of plasmin digestion. In contrast, both assays relying on the SCT were completely negative after 24 hours of plasmin digestion. All four methods yielded FSP titers of less than 10 micrograms/mL in 97 (78.2%) of 124 blood samples from normal donors. The SCT Sigma reagents consistently gave results of less than 10 micrograms/mL in all normal donors. No instance of an FSP value greater than 40 micrograms/mL was noted for the 124 normal donors. Of the 696 patient blood samples tested, the Dade assay gave the highest or equally highest (with respect to another FSP method) value in 604 (87%) cases; the Sigma SCT did so in 360 (52%); the TWT in 316 (45%); and the CBC assay in 184 (26%) cases. The Dade test classified the largest number of blood samples, 328 (47.1%), in the greater than 10 less than 40 micrograms/mL titer category; however, the proportion of cases (32.2%) in which this test yielded values greater than 40 micrograms/mL was about the same as those produced by the Sigma SCT (29.9%) and TWT products (25.8%). Thus, with the exception of the normal (less than 10 micrograms/mL) and the suspicious (10-40 micrograms/mL) range, all three methods (Dade, Sigma, and TWT) are comparable in their abilities to detect abnormal levels of FSP. In the normal range, the Dade method will yield results that are frequently in the suspicious range. The CBC was noticeably inferior in detecting both suspicious and frankly abnormal values of FSP. Eight patients with acute leukemia were monitored sequentially with FSP and fibrinopeptide A (FpA) assays during their first course of chemotherapy. In all instances, elevated FpA levels correlated with elevated FSP values, as determined by the Dade, Sigma, or TWT assays.(ABSTRACT TRUNCATED AT 400 WORDS)
Agglutination Tests↗