Plasma appearance and distribution of astaxanthin E/Z and R/S isomers in plasma lipoproteins of men after single dose administration of astaxanthin

Appearance, pharmacokinetics, and distribution of astaxanthin E/Z and R/S isomers in plasma and lipoprotein fractions were studied in 3 middle-aged male volunteers (37-43 years) after ingestion of a single meal containing a 100 mg dose of astaxanthin. The astaxanthin source consisted of 74% all-E-, 9% 9Z-, 17% 13Z-astaxanthin (3R,3'R-, 3R,3'S; meso-, and 3S,3'S-astaxanthin in a 1:2:1 ratio). The plasma astaxanthin concentration--time curves were measured during 72 hr. Maximum levels of astaxanthin (1.3 +/- 0.1 mg/L) were reached 6.7 +/- 1.2 hr after administration, and the plasma astaxanthin elimination half-life was 21 +/- 11 hr. 13Z-Astaxanthin accumulated selectively, whereas the 3 and 3'R/S astaxanthin distribution was similar to that of the experimental meal. Astaxanthin was present mainly in very low-density lipoproteins containing chylomicrons (VLDL/CM; 36-64% of total astaxanthin), whereas low-density lipoprotein (LDL) and high-density lipoprotein (HDL) contained 29% and 24% of total astaxanthin, respectively. The astaxanthin isomer distribution in plasma, VLDL/CM, LDL, and HDL was not affected by time. The results indicate that a selective process increases the relative proportion of astaxanthin Z-isomers compared to the all-E-astaxanthin during blood uptake and that astaxanthin E/Z isomers have similar pharmacokinetics.     

Analysis of omeprazole and 5-OH omeprazole in human plasma using hydrophilic interaction chromatography with tandem mass spectrometry (HILIC-MS/MS)--eliminating evaporation and reconstitution steps in 96-well liquid/liquid extraction

Bioanalytical methods using liquid/liquid extraction (LLE) and liquid chromatography with electrospray tandem mass spectrometry (LC-MS/MS) are widely used. The organic extracts need to be evaporated and reconstituted, hampering further improvement of throughput and automation. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well LLE by using hydrophilic interaction chromatography with MS/MS (HILIC-MS/MS) on silica column with high organic/low aqueous mobile phase. Omeprazole, its metabolite 5-OH omeprazole, and internal standard desoxyomeprazole, were extracted from 0.05 ml of human plasma using 0.5 ml of ethyl acetate in a 96-well plate. A portion (0.1 ml) of the ethyl acetate extract was diluted with 0.4 ml of acetonitrile and 10 microl was injected onto a Betasil silica column (50 mm x 3.0 mm, 5 microm) and detected by API 3000 and 4000 with (+) ESI. Mobile phase with linear gradient elution consists of acetonitrile, water, and formic acid (from 95:5:0.1 to 73.5:26.5:0.1 in 2 min). The flow rate was 1.5 ml/min with total run time of 2.75 min. The method was validated for a low limit of quantitation at 2.5 ng/ml for both analytes. The method was also validated for specificity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <4.4% relative standard deviation (R.S.D.) and 4.1% relative error (R.E.) for omeprazole, and 4.5% R.S.D. and 5.6% R.E. for 5-OH omeprazole, respectively.     

Specific radioimmunoassay of estrone sulfate. Application to measurement in male plasma

We described a new, specific and easy to use radioimmunoassay (RIA) of estrone sulfate (E1S) in males. After synthesis of an E1S-6-(O-carboxymethyl) oxime hapten then coupling to BSA, we obtained a specific anti-E1S antiserum. Although the cross-reactivity of DHEAS with our anti-E1S antiserum was low (CR=0.002%), we confirmed the absolute necessity of separating plasma DHEAS from plasma E1S, before E1S RIA, because in plasma, DHEAS is present at levels 3-6000-fold higher than E1S, which generally is ignored. Thus, we elicited an easy separation of DHEAS from E1S, by a fast chromatography on in-house minicolumns. This new RIA, was applied to the determination of E1S plasma normal values in males. In 27 young men (<35 years), mean+/-S.D. were 1.97nmol/l+/-1.07nmol/l and in 63 untreated healthy aged men (>55 years), 1.80nmol/l+/-1.21nmol/l. No significant difference was seen between young and older subjects. The ranges of E1S plasma levels in these subjects were rather large and the ratios between the highest and the lowest E1S plasma levels were seven in the young group and 23.4 in the older group. No decrease of E1S plasma levels was observed with ages. Contrary to large interindividual E1S plasma level variations, the intraindividual variations have been found to be no significant. Correlations between E1S and unconjugated estrogens, E2 and E1 were 0.22 (P=0.016) and 0.51 (0.001), respectively.     

Direct stereoselective assay of fluoxetine and norfluoxetine enantiomers in human plasma or serum by two-dimensional gas-liquid chromatography with nitrogen-phosphorus selective detection

A method was developed and validated for the direct enantioselective assay of fluoxetine and norfluoxetine in human plasma or serum by two-dimensional capillary gas-liquid chromatography (GC). A Rtx-1 fused-silica capillary (15 mx0.25 mm I.D., 1.0 micrometer film thickness) and a hydrodex-beta-6-TBDM fused-silica capillary (25 mx0.25 mm I.D., 0.25 micrometer film thickness) were used. A three-step liquid-liquid extraction was used for sample preparation with fluvoxamine and nisoxetine as internal standards. The method provided linear calibration between about 5 and 250 ng/ml for (R)- and (S)-fluoxetine as well as 15 and 250 ng/ml for (R)- and (S)-norfluoxetine. The limits of detection were about 1.5 and 6 ng/ml, respectively. Intra-day precision (coefficient of variation) was estimated as being between 5.4 and 12.7% at plasma levels of 25, 100 and 200 ng/ml for the four enantiomers. Inter-day precision was between 5.3 and 9.1% at 100 ng/ml. The enantioselective separation of some racemic psychopharmaceuticals was tested with various cyclodextrin GC-capillaries. Advantages and disadvantages of direct enantioselective GC are discussed for the assay of racemic psychopharmaceuticals. Samples from a patient who was treated with racemic fluoxetine were measured. In agreement with literature, plasma levels of the (R)-enantiomers of fluoxetine and norfluoxetine were considerably decreased in comparison to the (S)-enantiomers.     

Evidence for a dual action of converting enzyme inhibitor on blood pressure in normal man

We studied the effect of a converting enzyme inhibitor (CEI), Captopril SQ 14,225 50 mg p.o. in eight supine normal subjects under a high sodium (150 mEq/d) and low sodium (25 mEq/d) diet. On high sodium, plasma renin (PRA) and aldosterone were basal and Saralasin did not lower mean blood pressure. However, CEI induced an 11.4 +/- 3.2 mm fall in blood pressure (p less than 0.02) and either indomethacin 50 mg or ibuprofen 800 mg (PI), when given simultaneously on another day abolished the blood pressure response (2.5 +/- 0.9 mm Hg, p greater than 0.5). In contrast, on a low salt diet where renin was increased, CEI induced a drop in blood pressure which was not significantly altered by PI (12.8 +/- 1.1 vs. 10.0 +/- 3.1 mm Hg, p greater than 0.5). CEI increased plasma renin on both diets (1.7 +/- 0.5 to 3.5 +/- 0.8 and 2.8 +/- 0.6 to 12.5 +/- 3.1 ng/ml/hr respectively both p less than 0.05). Aldosterone did not change (high Na+) or fell (low Na+). Inhibition of Prostaglandin synthesis did not significantly block the renin rise from CEI suggesting that the direct angiotensin II negative feedback is relatively independent of acute prostaglandin release. Our studies suggest that CEI has a dual hypotensive action. In a low renin state, the hypotensive action appears to be mediated through vascular prostaglandins.     

Evidence from a dual action of converting enzyme inhibitor on blood pressure in normal man

We studied the effect of a converting enzyme inhibitor (CEI), Captopril SQ 14,225 50 mg p.o. in eight supine normal subjects under a high sodium (150 meq/d) and low sodium (25 mEq/d) diet. On high sodium, plasma renin (PRA) and aldosterone were basal and Saralasin did not lower mean blood pressure. However, CEI induced an 11.4±3.2 mm fall in blood pressure (p<0.02) and either indomethacin 50 mg or ibuprofen 800 mg (PI), when given simultaneously on another day, abolished the blood pressure response (2.5±0.9 mm Hg, p>0.5). In contrast, on a low salt diet where renin was increased, CEI induced a drop in blood pressure which was not significantly altered by PI (12.8±1.1 vs. 10.0±3.1 mm Hg, p>0.5). CEI increased plasma renin on both diets (1.7±0.5 to 3.5±0.8 and 2.8±0.6 to 12.5±3.1 ng/ml/hr respectively both p<0.05). Aldosterone did not change (high Na⁺) or fell (low Na⁺). Inhibition of prostaglandin synthesis did not significantly block the renin rise from CEI suggesting that the direct angiotensin II negative feedback is relatively independent of acute prostaglandin release. Our studies suggest that CEI has a dual hypotensive action. In a low renin state, the hypotensive action appears to be mediated through vascular prostaglandins.     

Stereoselective disposition of tiaprofenic acid enantiomers in rats

The pharmacokinetics and metabolic chiral inversion of the S(+)‐ and R(−)‐enantiomers of tiaprofenic acid (S‐TIA, R‐TIA) were assessed in vivo in rats, and in addition the biochemistry of inversion was investigated in vitro in rat liver homogenates. Drug enantiomer concentrations in plasma were investigated following administration of S‐TIA and R‐TIA (i.p. 3 and 9 mg/kg) over 24 hr. Plasma concentrations of TIA enantiomers were determined by stereospecific HPLC analysis. After administration of R‐TIA it was found that 1) there was a time delay of peak S‐TIA plasma concentrations, 2) S‐TIA concentrations exceeded R‐TIA concentrations from ∼2 hr after dosing, 3) C_max and AUC_(0‐∞) for S‐TIA were greater than for R‐TIA following administration of S‐TIA, and 4) inversion was bidirectional but favored inversion of R‐TIA to S‐TIA. Bidirectional inversion was also observed when TIA enantiomers were incubated with liver homogenates up to 24 hr. However, the rate of inversion favored transformation of the R‐enantiomer to the S‐enantiomer. In conclusion, stereoselective pharmacokinetics of R‐ and S‐TIA were observed in rats and bidirectional inversion in rat liver homogenates has been demonstrated for the first time. Chiral inversion of TIA may involve metabolic routes different from those associated with inversion of other 2‐arylpropionic acids such as ibuprofen. Chirality 11:103–108, 1999. © 1999 Wiley‐Liss, Inc.     

A liquid chromatographic-tandem mass spectrometric method for the determination of two selective thymidylate synthase inhibitors, BGC945 and BGC638, in mouse plasma

A LC-tandem mass spectrometry method to quantify the quinazoline-based thymidylate synthase inhibitors BGC945 and BGC638 in mouse plasma was developed. BGC945 and BGC638 were extracted from mouse plasma using protein precipitation with acetonitrile. Chromatography was performed on a Fluophase RP 5 microm, 100 mmx2.0mm i.d. column using a gradient of ammonium acetate and acetonitrile as a mobile phase with a flow rate of 0.2 mLmin(-1). The injection volume for each sample was 20 microL with a total run time of 7.5 min. This method was validated in the range 25-4000 nM (r2=0.99). The analytical assay performance showed that the method was accurate (mean intra- and inter-day assay R.E. were below 12% and 11%, respectively), reproducible (mean intra- and inter-day R.S.D. were less than 13% and 5% for all quality control levels, respectively) and sensitive (lower limit of quantification was 25 nM) in the range studied. This validated method has been used to define the first pharmacokinetic report of BGC945 and BGC638 in mice.     

Effect of exercise to rest ratio on plasma lactate concentration at work rates above and below maximum oxygen uptake

The metabolic and physiological responses to different exercise to rest ratios (E:R) (2:1, 1:1, 1:2) of eight subjects exercising at work rates approximately 10% above and below maximum oxygen uptake (VO2max) were assessed. Each of the six protocols consisted of 15 1-min-long E:R intervals. Total work (kJ), oxygen uptake (VO2), heart rate (fc) and plasma lactate concentrations were monitored. With increases in either E:R or work rate, VO2 and fc increased (P < 0.05). The average (15 min) VO2 and fc ranged from 40 to 81%, and from 62 to 91% of maximum, respectively. Plasma lactate concentrations nearly doubled at each E:R when work rate was increased from 90 to 110% of VO2max and ranged from a low of 1.8 mmol.l-1 (1:2-90) to a high of 10.7 mmol.l-1 (2:1-110). The 2:1-110 protocol elicited plasma lactate concentrations which were approximately 15 times greater than that of rest. These data suggest that plasma lactate concentrations during intermittent exercise are very sensitive to both work rate and exercise duration.     

An efficient system for the asymmetric acylation of (R,S)-3-n-butylphthalide catalyzed by novozyme 435

Novozyme 435 could be a highly efficient catalyst in the asymmetric acylation of (R,S)-3-n-butylphthalide in tetrahydrofuran-hexane solvents. The effect of various reaction parameters such as agitation velocity, water content, mixed media, temperature, concentration of Novozyme 435, molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, reaction time, enantiomeric excess of substrate (ee(S)), enantiomeric excess of product (ee(P)), and enantioselective ratio (E) were studied. Tetrahydrofuran markedly improved (R,S)-3-n-butylphthalide conversion, enantiomeric excess of remaining 3-n-butylphthalide, and enantiomeric ratio. The optimum media were 50% (v/v) tetrahydrofuran and 50% (v/v) hexane. Other ideal reaction conditions were an agitation velocity of 150 rpm, 0.4% (v/v) water content, temperature of 30 °C, 8 mg/mL dosage of Novozyme 435, 8:1 (0.4 mmol: 0.05 mmol) molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, and a reaction time of 48 hr. Under the optimum conditions, 96.4% ee(S) and 49.3% conversion of (R,S)-3-n-butylphthalide were achieved. In addition, enantiomeric excess of the product was above 98.0%.     

Subpopulations of chicken B lymphocytes.

Immunoglobulin-synthesizing cells from the spleen and bursa were fractionated by the 1 X G sedimentation velocity technique and characterized by their ability to synthesize immunoglobulin and by staining with fluorescent anti-light chain chain. Four subpopulations of immunoglobulin-synthesizing cells were identified. In the bursa, slowly sedimenting (S 2.3 mm/hr) and rapidly sedimenting (S greater than 3.5 mm/hr) subpopulations with surface immunoglobulin were present; in the spleen, a slowly sedimenting (S 2.3 mm/hr) subpopulation with surface immunoglobulin and plasma cells (S greater than 3.5 mm/hr) with large concentrations of intracellular immunoglobulin existed. The subpopulations differed most markedly in their ability to synthesize immunoglobulin (cpm Ig synthesized/10(6) Ig-positive cells); the rates of immunoglobulin synthesis were in the ratio 1:2:1:900. The slowly sedimenting B cells from the spleen and both subpopulations of B cells from the bursa released small amounts of immunoglobulin into the culture media, whereas, the plasma cells released immunoglobulin at a rate as much as 3700 times greater. Bursal B cells could be further distinguished from splenic B cells by a greater amount of DNA synthesis.     

Determination of a novel substance P inhibitor in human plasma by high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometric detection using single and triple quadrupole detectors

Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2R)-[1(R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3(S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant (I) and internal standard (II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid-liquid extraction with methyl-t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific's Javelin BDS C-8 2 mm x 4.6 mm 3 microm guard column coupled to BDS C-8 50 mm x 4.6 mm 3 microm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor --> ion combinations of m/z 535 --> 277 and 503 --> 259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.     

Cobalt substitution of mouse R2 ribonucleotide reductase as a model for the reactive diferrous state: spectroscopic and structural evidence for a ferromagnetically coupled dinuclear cobalt cluster

The R2 dimer of mouse ribonucleotide reductase contains a dinuclear iron-oxygen cluster and tyrosyl radical/subunit. The dinuclear diferrous form reacts with dioxygen to generate the tyrosyl radical essential for the catalytic reaction that occurs at the R1 dimer. It is important to understand how the reactivity toward oxygen is related to the crystal structure of the dinuclear cluster. For the mouse R2 protein, no structure has been available with a fully occupied dinuclear metal ion site. A cobalt substitution of mouse R2 was performed to produce a good model for the very air-sensitive diferrous form of the enzyme. X-band EPR and light absorption studies (epsilon(550 nm) = 100 mm(-1) cm(-1)/Co(II)) revealed a strong cooperative binding of cobalt to the dinuclear site. In perpendicular mode EPR, the axial signal from mouse R2 incubated with Co(II) showed a typical S = 3/2 Co(II) signal, and its low intensity indicated that the majority of the Co(II) bound to R2 is magnetically coupled. In parallel mode EPR, a typical integer spin signal (M(s) = +/-3) with g approximately 12 is observed at 3.6 K and 10 K, showing that the two Co(II) ions (S = 3/2) in the dinuclear site are ferromagnetically coupled. We have solved the 2.4 A crystal structure of the Co(II)-substituted R2 with a fully occupied dinuclear cluster. The bridging Co(II) carboxylate ligand Glu-267 adopts an altered orientation compared with its counterpart Glu-238 in Escherichia coli R2. This might be important for proper O(2) activation of the more exposed native diferrous site in mouse R2 compared with E. coli R2.     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

Conjugation reactions catalyzed by bifunctional proteins related to beta-oxidation in bile acid biosynthesis

The conjugation reactions of hydration and dehydrogenation catalyzed by the dehydratase and dehydrogenase activities of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (DBP) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein (LBP) in the side chain degradation step of bile acid biosynthesis were investigated using chemically synthesized C27-bile acid CoA esters as substrates. The hydration catalyzed by DBP showed high diastereoselectivity for (24E)-3alpha,7alpha,12alpha-trihydroxy- and (24E)-3alpha,7alpha-dihydroxy-5beta-cholest-24-en-26-oyl CoA to give (24R,25R)-3alpha,7alpha,12alpha,24-tetrahydroxy- and (24R,25R)-3alpha,7alpha,24-trihydroxy-5beta-cholestan-26-oyl CoAs, respectively, and the dehydrogenation catalyzed by DBP also showed high stereospecificity for the above (24R,25R)-isomers to give 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-24-oxo-5beta-cholestan-26-oyl CoAs, respectively. On the other hand, the dehydratase activity of LBP displayed a different diastereoselectivity producing the (24S,25S)-isomer, and dehydrogenase activity of LBP was stereospecific for the (24S,25R)-isomer to give the above 24-oxo-derivative. The hydration and dehydrogenation reactions catalyzed by DBP were effectively conjugated to convert (24E)-5beta-cholestenoyl CoA to 24-oxo-5beta-cholestanoyl CoA. However, the reactions catalyzed by LBP were not conjugated. These results indicate that DBP plays an important role in the biosynthesis of bile acid.     

Responsiveness of plasma aldosterone to angiotensin II in patients with diabetes mellitus

Aldosterone responsiveness to angiotensin II (A II) was evaluated in 65 diabetic patients with and without various diabetic complications versus 38 age-matched non-diabetic subjects. Plasma aldosterone (PA), together with plasma renin activity (PRA), was low and responded poorly to furosemide (80 mg, orally) plus upright posture (4 hours) stimulation in diabetic patients. When the PA response to stimulation relative to PRA response was estimated from the ratio of PA increase to PRA increase after stimulation (delta PA/delta PRA), the 38 non-diabetic subjects had ratios more than 3.0. Of the 65 diabetic patients, 48 had normal delta PA/delta PRA ratios (more than 3.0) and 17 had low delta PA/delta PRA ratios (less than 2.9). Graded A II infusions (1, 2, and 4 ng/kg/min each for 30 min) were performed under a low sodium intake (sodium, 120 mEq/day) in 25 of the 65 diabetic patients, whose delta PA/delta PRA ratios were normal in 15 and low in 10, and in 16 non-diabetic subjects. The PA responses to the graded A II infusions in the normal delta PA/delta PRA diabetic patients were similar to those in the non-diabetic subjects. However, the PA responses to the graded A II infusions in the low delta PA/delta PRA diabetic patients were significantly lower. It is concluded that, although the majority of diabetic patients have normal aldosterone responsiveness to A II, some diabetic patients have blunted aldosterone responsiveness to A II probably attributable to the abnormality of the adrenal cortex in addition to the impaired renin secretion.     

Adaptation to Cd(II) and Zn(II), and the caffeine-potentiated override of the G2 block induced by the checkpoint activated by DNA damage

ABSTRACT

Moderate and low concentrations of Zn(II) and Cd(II) were defined as those which depressed the rate of root elongation in Allium cepa L. to about 40 and 70% respectively of the control (17.3 ± 4.9 mm/day) at 25°C. At moderate concentrations, cells were detoxified from Cd(II), but not from Zn(II), by inducing the heavy metal chelators phytochelatins. Thus, root elongation further decreased (from 41 to 19% of the control) at moderate (0.05 mM) Cd(II) concentration upon addition of 0.25 mM L-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of phytochelatin synthesis. On the other hand, cells were also detoxified from Zn(II) by an alternative mechanism, as the 42% inhibition displayed at 0.5 mM Zn(II) concentration was partially reversed (up to 79%) in the presence of BSO. Zn(II) activated the checkpoint pathway induced by DNA damage, as a transient G₂ block was produced; this block was partially cancelled by caffeine, so that chromosomal bridges (but no breaks) were observed in ana-telophase. On the other hand, Cd(II) did not activate the DNA damage checkpoint, as cells entered into anaphase with chromosomal breaks and bridges without any delay. Cd(II) may preclude the recognition of DNA damage by altering protein-DNA interactions, since 30% of the metaphases displayed clumped chromosomes. A minimum threshold was required to induce the adaptive responses described here, as BSO did not modify the reduction in root elongation rate recorded at low concentrations of both heavy metals.     

Yaba tumor poxvirus synthesis in vitro. II. Adsorption, inactivation, and assay studies

Yohn, David S. (Roswell Park Memorial Institute, Buffalo, N.Y.), Fanny R. Marmol, Victoria A. Haendiges, and James T. Grace, Jr. Yaba tumor poxvirus synthesis in vitro. II. Adsorption, inactivation, and assay studies. J. Bacteriol. 91:1953-1958. 1966.-Means to increase the efficiency of the Yaba tumor poxvirus assay in BSC-1 cell cultures were sought. A method was devised wherein 0.4 ml of each virus dilution was layered onto BSC-1 cells in 80-mm Leighton tubes and permitted to adsorb at 25 C for 18 hr prior to incubation at 35 C. The diluent found most reliable was medium 199 containing 50% bovine amniotic fluid adjusted to 2.0 mm calcium and 1.0 mm magnesium at pH 7.0. These modifications yielded highly reproducible titrations with a greater than twofold increase in assay sensitivity. Irreversible adsorption of Yaba virus by BSC-1 cells proceeds comparatively slowly at 25 to 33 C. Although the process is more rapid at 35 or 37 C, the increased thermal lability of the virus at these temperatures results in lower titers than with virus adsorbed at 25 C for longer periods of time. Yaba poxvirus appears to be 5 to 10 times more sensitive to thermal inactivation than vaccinia poxvirus.     

Cerebrospinal fluid S100B is elevated in the earlier stages of Alzheimer's disease

Postmortem demonstration of increased expression of biologically active S100B in Alzheimer's disease (AD) and its relation to progression of neuropathological changes across the cortical regions suggests involvement of this astrocytic cytokine in the pathophysiology of AD. The hypothesis that the overexpression of S100B in Alzheimer brain is related to the progression of clinical symptoms was addressed in living persons by measuring S100B concentrations in cerebrospinal fluid (CSF) from AD patients with a broad range of clinical dementia severity and from healthy older persons. The effect of normal aging on CSF S100B concentrations also was estimated. CSF S100B did not differ between all 68 AD subjects (0.98±0.09 ng/ml (mean±S.E.M.)) and 25 healthy older subjects (0.81±0.13 ng/ml). When AD subjects were divided into mild/moderate stage and advanced stage clinical dementia severity by the established Clinical Dementia Rating Scale (CDR) criteria, S100B was significantly higher in the 46 mild/moderate stage AD subjects (1.17±0.11 ng/ml) than in either the 22 advanced stage AD subjects (0.60±0.12 ng/ml) or the healthy older subjects. Consistent with higher CSF S100B in mild to moderate AD, there was a significant correlation among all AD subjects between CSF S100B and cognitive status as measured by the Mini Mental State Exam (MMSE) score. CSF S100B did not differ between healthy older subjects and healthy young subjects. These results suggest increased CNS expression of S100B in the earlier stages of AD, and are consistent with a role for S100B in the initiation and/or facilitation of neuritic plaque formation in AD brain.     

Enantioselective binding of mephobarbital to plasma proteins

The enantioselective protein binding of mephobarbital (MPB) was investigated in human plasma and human serum albumin solutions by equilibrium dialysis. A small but statistically significant difference was observed in the in vitro plasma protein binding of the enantiomers; (S)-MPB was approximately 59% bound and (R)-MPB approximately 67% bound. The binding to albumin [(S)-MPB: approximately 29% bound, and (R)-MPB: approximately 41% bound] was less than to plasma proteins but showed somewhat greater enantioselectivity, suggesting that albumin binding is a major source of the enantioselectivity in plasma. The effects of MPB concentration, of varying enantiomeric concentration ratio, and of phenobarbital on the enantioselective binding of MPB were studied. The effect of age was also investigated by measuring the binding in plasma from 8 young (18-25 yr) and 8 elderly (greater than 60 yr) male subjects who took single doses of MPB. The results were in close agreement with the in vitro binding data, and the binding of both enantiomers was marginally but significantly lower in the young compared with the elderly subjects. These differences in binding were consistent with previously observed pharmacokinetic differences between the two subject groups.