Biomedical subjects
Publications and source records attributed to F M Chen.
A bifunctional ligand, 6-(5-carboxyl pentyl)-5,7 dioxo-1,4,8, 11-tetraazaundecane, has been shown to produce a neutral, inert complex with Pd(II). The complex is formed in high yield and is stable over a wide pH range. It can be conjugated to proteins through the carboxylic acid side arm using a carbodiimide coupling reagent and may be useful for labeling antibodies or other large molecules.
DNA covalent binding studies with enantiomers of trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (anti-BPDE) have been carried out by means of spectroscopic techniques (UV, CD, and fluorescence). Synthetic polynucleotides are employed to investigate binding differences between the G.C and A.T base pairs and to elucidate the bases for the stereoselective covalent binding of DNA toward anti-BPDE. The results indicate that of all the polynucleotides studied, only poly(dA-dT).poly(dA-dT) exhibits predominant intercalative covalent binding towards (+)-anti-BPDE and suffers the least covalent modification. Only minor intercalative covalent contributions are found in alternating polymer poly(dA-dC).poly(dG-dT). These observations parallel the DNA physical binding results of anti-BPDE and its hydrolysis products. They support the hypothesis that intercalative covalent adducts derive from intercalative physical binding while the external covalent adducts derive from external bimolecular associations. In contrast to the A.T polymers, the guanine containing polymers exhibit pronounced reduction in covalent modification by (-)-anti-BPDE. The intercalative covalent binding mode becomes relatively more important in the adducts formed by the (-) enantiomer as a consequence of decreased external guanine binding. These findings are consistent with the guanine specificity, stereoselective covalent binding at dG, the absence of stereoselectivity at dA for anti-BPDE, and the enhanced binding heterogeneity for the (-) enantiomer as found in the native DNA studies. The possible sequence and/or conformational dependence of such stereoselective covalent binding is indicated by the opposite pyrenyl CD sign exhibited by (+)-anti-BPDE bound to polynucleotides with pyrimidine on one strand and purine on another vs. that bound to polymers containing alternating purine-pyrimidine sequences.
A procedure is described for the synthesis of crystalline N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts starting from the commercially available N2-tert-butoxycarbonyl-N6-benzyloxycarbonyl-L-lysine, which is reacted with methyl iodide in methanol in the presence of potassium hydrogen carbonate after deprotection of the side-chain amino group by catalytic hydrogenation. The work-up involves only filtrations and evaporations.
Circular dichroism (CD) as well as absorption spectral measurements reveals that poly(dG-m5dC).poly(dG-m5dC) suffers more extensive covalent modification by (+)-dihydroxy-anti-epoxybenzo[a]pyrene [(+)-anti-BPDE] than its unmethylated counterpart and that the covalently attached pyrenyl moiety exhibits stronger stacking interactions with the bases in the methylated polymer as suggested by the much larger pyrenyl spectral red shifts, most likely the consequence of intercalation. Stereoselective binding properties of these polymers are evidenced by the much reduced preference for the (-) enantiomer. Modifications due to (+)-anti-BPDE on the 50 microM hexaamminecobalt induced Z DNAs are much less pronounced and much less stereoselective, with the pyrenyl spectral characteristics being distinct from those of the B form. Salt titrations on the (+)-anti-BPDE modified poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) indicate much reduced cooperativity on the B to Z transition when compared to the unmodified counterparts. Evidence also suggests that covalent modification by anti-BPDE inhibits the B to Z conversion of base pairs in its immediate vicinity, presumably through intercalative stabilization of the B conformer at high salt. In contrast to stabilizing the B conformation for the proximal base pairs, covalent lesion by (+)-anti-BPDE appears to destabilize distal base pairs with the consequence of kinetic facilitation of B to Z transformation for these regions. Interesting differential effects on the reverse Z to B transforming abilities of these two enantiomers are observed with the covalent binding of the (-) isomer showing higher potency for inducing such conversion.
Spectroscopic studies on the trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene- (anti-BPDE-) modified synthetic polynucleotide solutions reveal interesting sequence-dependent stereoselective covalent binding of anti-BPDE to DNA. Absorption spectral results indicate that the G.C polymers are much more reactive than the A.T polymers toward this metabolite and the homopolymer suffers higher modification than its corresponding alternating polynucleotide. The covalently attached anti-BPDE exhibits only a 2-3-nm red shift in the guanine-containing polynucleotide and native DNA solutions as opposed to the 8-nm red shift in poly(G) and none in the A.T polymers. Distinct stereoselectivities are exhibited by poly(dG-dC).poly(dG-dC) vs. poly(dG).poly(dC) as suggested by the oppositely signed CD in the pyrene spectral region. Comparison with the syn-BPDE modified polynucleotides reveals some interesting differences with its anti diastereomer. Significant contributions from the intercalated syn-BPDE are apparent in the modified guanine-containing polynucleotides as indicated by the appearance of 10-nm red-shifted shoulders. In contrast to the strong dependence on polynucleotides for anti-BPDE, the rate of hydrolysis of syn-BPDE appears to be insensitive to their presence in the solution. anti-BPDE modification on the 50 microM hexaamminecobalt-induced Z-form poly(dG-dC).poly(dG-dC) is much less extensive than its corresponding B form, possibly the consequence of both structural and ionic strength factors. The spectral characteristics of anti-BPDE bonded to these two forms are distinctly different, with the Z form resembling more closely those of A.T polymers.(ABSTRACT TRUNCATED AT 250 WORDS)
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Comparative studies on the salt titration and the related kinetics for poly(dG-dC) X poly(dG-dC) in pH 7.0 and 3.8 solutions clearly suggest that base protonation facilitates the kinetics of B-Z interconversion although the midpoint for such a transition in acidic solution (2.0-2.1 M NaCl) is only slightly lower than that of neutral pH. The rates for the salt-induced B to Z and the reverse actinomycin D induced Z to B transitions in pH 3.8 solutions are at least 1 order of magnitude faster than the corresponding pH 7.0 counterparts. The lowering of the B-Z transition barrier is most likely the consequence of duplex destabilization due to protonation as indicated by a striking decrease (approximately 40 degrees C) in melting temperature upon H+ binding in low salt. The thermal denaturation curve for poly(dG-dC) X poly(dG-dC) in a pH 3.8, 2.6 M NaCl solution indicates an extremely cooperative melting at 60.5 degrees C for protonated Z DNA, which is immediately followed by aggregate formation and subsequent hydrolysis to nucleotides at higher temperatures. The corresponding protonated B-form poly(dG-dC) X poly(dG-dC) in 1 M NaCl solution exhibits a melting temperature about 15 degrees C higher, suggesting further duplex destabilization upon Z formation.
Comparative binding studies of tetraols (anti- BPTs ) derived from trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetra- hydrobenzo [a]py rene (anti-BPDE) with native DNA in neutral and acidic solutions reveal that 4-5 times stronger intercalative binding occurs in the protonated duplex DNA than in the unprotonated one. Spectroscopic (absorption and circular dichroism) investigations with synthetic polynucleotides indicate that in neutral pH, anti- BPTs intercalate significantly in poly(dA-dT):poly(dA-dT), only slightly in guanine containing alternating polynucleotides poly(dG-dC): poly(dG-dC) and poly(dA-dC):poly(dG-dT), and hardly in homopolymers poly(dG):poly(dC) and poly(dA):poly(dT). In acidic solutions, on the other hand, all three alternating polynucleotides, poly(dG-dC):poly(dG-dC) in particular, exhibit strong intercalative binding to anti- BPTs although the homopolymers still lack such capability. These results are very similar to those of corresponding pyrene studies suggesting that pyrene can be a useful model compound for the DNA binding studies of benzo[a]pyrene metabolites. The observed enhanced binding of anti- BPTs in the acidic natural DNA solution is seen as the consequence of changes in base sequence preference upon base protonation.
Solubilization as well as spectral studies of pyrene in natural DNA and synthetic deoxypolynucleotide solutions at neutral pH reveal at least two binding modes. Sites I are predominant in native DNA and in poly(dA-dT): poly(dA-dT) whereas sites II are found with denatured DNA and other polynucleotides such as poly(dA):poly(dT) and three different types of guanine containing copolymers which solubilize pyrene to a lesser extent. Spectral comparison with the covalent adducts of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydro-benzo(a)pyrene (anti-BPDE) and the physical complexes of its tetraols lead to the suggestion of a base sequence specific binding model for this carcinogenic metabolite to account for the puzzling fact that although its physical binding is predominantly intercalative, the covalent adducts appear not to be intercalated. It is speculated that in neutral solutions, intercalation may have little, if any, to do with the chemical lesion of this metabolite to the guanine base of the DNA and may, on the contrary, provide an efficient pathway for detoxification.
A simple method for detecting the amount of a polycyclic aromatic hydrocarbon (PAH) solubilized in DNA solutions is described. This method takes advantage of the fact that sodium dodecyl sulfate (SDS) induces dissociation of PAH from DNA, and the greatly enhanced solubility of PAH in SDS solution makes possible the direct preparation of a standard solution for the extinction coefficient determination. The ability to make direct comparison between the spectroscopic characteristics before and after dissociation is accomplished by utilizing a tandem cuvette. The usefulness of this technique is then demonstrated by applying it to solubilization studies of pyrene in native and denatured DNA solutions of varying sodium chloride concentrations as well as in deoxymononucleotide solutions. Binding constants are estimated and the solubility data for pyrene in the DNA solutions are interpreted in terms of the binding abilities of intercalative versus external sites and the effect of salt. The mononucleotide results suggest a binding preference of pyrene to purine bases. It was observed that the binding constant of mononucleotide to pyrene is an order of magnitude lower than that of single-stranded DNA, which in turn is an order of magnitude smaller than that of duplex DNA.
Chronic infusion of angiotensin II (ANG II) into the olfactory bulb (OB) elicited a moderate dipsogenesis which occurred only during the dark phase, essentially doubling the water-to-food intake ratio. Removal of the food from the ANG II-OB group reduced water consumption to the level of the saline-infused/food-deprived controls. These experiments suggest that ANG II may interact with the OB to alter the normal water/food relationship.
Last year, after a systematic search of pathological and patho-physiological changes of inhalation injury in dogs, we recorded severe pulmonary oedema as early as 2 h postburn (the earliest monitoring time designed in experiment) on dogs inflicted with both body surface and severe respiratory burns without any fluid replacement during the whole course of experiment. In an analysis of cases admitted into our burn centre in the past ten years, we also found that there was no definite relationship between the incidence and severity of pulmonary oedema occurring in severe burn patients and the amount of fluid infused in the early burn phase. These facts led us to the following experiment designed to study the effect of intravenous infusion on development of pulmonary oedema after severe burns complicated with inhalation injuries.
Planar pyrene molecules can acquire optical activity upon binding to the disymmetric environment of duplex DNA. Positive induced Cotton effects are observed for pyrene in basic and neutral DNA solutions but revert to strongly negative CD bands in moderately acidic solutions. pH titrations indicate that this change over is strongly cooperative until acid denaturation sets in. The negative induced CD for pyrene results from its binding to a protonated duplex state which shows a striking 40 degrees C decrease in the melting temperature from its neutral counterpart with 0.18 M NaCl. Further studies with synthetic polynucleotides reveal that pyrene strongly prefers the guanine containing sequences in general and dG-dC (and/or dC-dG) sequence(s) in particular for this protonated duplex state, in distinct contrast to the dA-dT (and/or dT-dA) sequence specificity in the neutral solutions. The observed pyrene CD sign reversal upon protonation is thus the manifestation of DNA conformational change and the accompanied alteration in base sequence specificity.
We have demonstrated previously that angiotensin II (ANG II) receptors in the rat pituitary fluctuate with the stage of the estrous cycle, with the highest binding level found in diestrus and the lowest found in estrus. These findings were extended in the present study by examining the role of estrogens in variations of ANG II binding. Ovariectomized rats were treated with a Silastic implant containing estradiol benzoate for 4 or 14 days before death, and pituitary [125I]ANG II binding was measured. Compared to that in ovariectomized controls, ANG II binding in the anterior pituitary was found to be significantly reduced after either 4 or 14 days of estrogen treatment. This reduction was mainly an effect on receptor density rather than receptor affinity. The observed apparent Kd and receptor density were as follows: ovariectomized controls, 0.8 nM and 92 fmol/mg protein; 4 days of estrogen treatment, 1.0 nM and 25 fmol/mg protein; 14 days of estrogen treatment, 0.8 nM and 12 fmol/mg protein. We conclude that estrogen treatment leads to a reduction in ANG II-binding sites in the anterior pituitary, and this effect may account for the reduction of pituitary ANG II receptors at estrus.
Perturbation of peripheral volume homeostasis results in regionally selective changes in brain angiotensinogen, the putative precursor of angiotensin II. The results presented indicate that brain angiotensinogen may be affected in a selective and regional manner and that steroid hormones may mediate these changes. Sex and adrenal steroids appear to affect angiotensinogen levels in different areas of the rat brain. Brain angiotensinogen and norepinephrine levels are linearly and positively correlated. Reserpinization leads to selective increases in brain angiotensinogen which are shown to be attributable to adrenal corticosterone secretion. These results point to steroids as messengers controlling in part the level of activity of brain catecholaminergic and peptidergic systems which regulate the peripheral cardiovascular system.
The peptide hydrazides of N-benzyloxycarbonylglycyl-X where X = alanine, leucine, phenylalanine, valine and isoleucine have been prepared by hydrazinolysis of the corresponding methyl esters and 2,4-distributed-5 (4H)-oxazolones, and by the carbodiimide-mediated reactions of the peptide acids with hydrazine in the presence of 1-hydroxybenzotriazole. The stereochemical purity of the products was examined by coupling them by the azide method with an ester of lysine followed by analysis for the diastereomeric tripeptides by an established procedure. The results concur with and substantiate the generally held notions on the chiral stability associated with hydrazinolysis and the azide procedure, except that an optically pure hydrazide could not be obtained from the oxazolone of the alanyl peptide.
The effects of continuous infusion of angiotensin II (ANG II, 1 microgram hr-1) into the olfactory bulb (O.B.) were studied on rats with chronically indwelling intracerebral cannulae. Chronic infusion (ALZA minipump system) of ANG II into the O.B. elicited a moderate dipsogenesis as compared to saline infused animals. The increased drinking occurred only during the dark phase (6 pm - 6 am) and appeared to be associated with food intake, which also occurred during this time. Removal of the food or disconnection of the minipump reduced water consumption to comparable levels in both groups. Acute injection of ANG II (250 ng) into the O.B. failed to elicit drinking. Since the olfactory bulb has a high concentration of ANG II receptors, these experiments suggest that ANG II may interact with the O.B. to facilitate the drinking associated with food intake.