Involvement of prostaglandin E2 in production of amyloid-beta peptides both in vitro and in vivo

Amyloid-beta peptides (Abeta), generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE(2) receptors (EP(1-4)), EP(4) receptors alone or EP(2) and EP(4) receptors together are responsible for this PGE(2)-stimulated production of Abeta in HEK293 or SH-SY5Y cells, respectively. An EP(4) receptor antagonist suppressed the PGE(2)-stimulated production of Abeta in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Abeta, demonstrating that increases in the cellular level of cAMP are responsible for the PGE(2)-stimulated production of Abeta. Immunoblotting experiments and direct measurement of gamma-secretase activity suggested that PGE(2)-stimulated production of Abeta is mediated by activation ofgamma-secretase but not of beta-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Abeta in the brain, when they were crossed with mice lacking either EP(2) or EP(4) receptors, suggesting that PGE(2)-mediated activation of EP(2) and EP(4) receptors is involved in the production of Abeta in vivo and in the pathogenesis of AD.     

Importance of guanine nitration and hydroxylation in DNA in vitro and in vivo

Guanine (Gua) modification by nitrating and hydroxylating systems was investigated in DNA. In isolated calf thymus DNA, 8-NO(2)-Gua and 8-oxo-Gua were dose-dependently formed with peroxynitrite, and 8-NO(2)-Gua was released in substantial amounts. Myeloperoxidase (MPO) with H(2)O(2) and NO(2)(-) reacted with calf thymus DNA to form 8-NO(2)-Gua dose dependently without release of 8-NO(2)-Gua. The frequency of strand breaks was higher than the sum of 8-NO(2)-Gua and 8-oxo-Gua, particularly in the MPO-treated DNA, indicating the importance of other types of damage. The activation of human neutrophils and lymphocytes with phorbol ester did not induce 8-NO(2)-Gua and 8-oxo-Gua in their nuclear DNA. However, 8-NO(2)-Gua was found in calf thymus DNA co-incubated with activated neutrophils in the presence of NO(2)(-). No significant formation of 8-NO(2)-Gua was found in liver DNA from mice treated with Escherichia coli lipopolysaccharide. The incubation of peroxynitrite or MPO-H(2)O(2)-NO(2)(-)-treated DNA with formamidopyrimidine glycosylase (Fpg) released 8-oxo-Gua, but not 8-NO(2)-Gua, indicating that 8-NO(2)-Gua is not a substrate for Fpg. Although 8-NO(2)-Gua was generated in isolated DNA by different nitrating systems, other types of damage were formed in abundance, and the lesion could not be found reliably in nuclear DNA, suggesting that the biological importance is limited.     

Involvement of peripheral type of benzodiazepine receptor in social isolation stress-induced decrease in pentobarbital sleep in mice

Our previous studies have shown that central-type benzodiazepine (BZD) receptors (CBR) and neurosteroids capable of modulating GABA(A) receptor function are involved in the decrease of pentobarbital (PB)-induced sleep caused by social isolation stress in mice. In this study, to further clarify the mechanism underlying this decrease, we investigated the possible involvement of peripheral-type BZD receptors (PBR) which play an important role in neurosteroidogenesis in PB sleep in socially isolated mice. Socially isolated mice showed significantly shorter duration of PB-induced sleep than group-housed animals. When injected intracerebroventricularly (i.c.v.), FGIN-1-27 (FGIN, 25-100 nmol), a selective PBR agonist, and PK11195 (PK, 14-28 nmol), a PBR antagonist, and pregnenolone (PREG, 15-30 nmol), a neurosteroid precursor, dose-dependently normalized the PB sleep in isolated mice without having an effect on the group-housed animals. In contrast, pregnenolone sulfate (PS, 24 nmol), an endogenous neurosteroidal negative allosteric modulator of the GABA(A) receptor, reduced PB sleep in group-housed but not isolated mice. PS, at the same dose, significantly attenuated the effects of FGIN (100 nmol), PK (28 nmol) and PREG (30 nmol) in isolated mice, while FGIN (100 nmol), PK (28 nmol) and pregnenolone (30 nmol) significantly blocked the effect of PS (24 nmol) in group-housed mice. These results suggest that the PBR-mediated decrease in the genesis of neurosteroid(s) possessing a GABA(A) receptor agonistic profile is also partly involved in the down regulation of the GABA(A) receptor following long-term social isolation and contributes to the decrease of PB-induced sleep in isolation stressed mice.     

Stereoselective metabolism of pentoxifylline in vitro and in vivo in humans

Pentoxifylline increases erythrocyte flexibility, reduces blood viscosity, and inhibits platelet aggregation and is thus used in the treatment of peripheral vascular disease. It is transformed into at least seven phase I metabolites, of which two, M1 and M5, are active. The reduction of the keto group of pentoxifylline to a secondary alcohol in M1 takes place chiefly in erythrocytes, is rapidly reversible, and creates a chiral center. The aims of this study were: to develop HPLC methods to separate the enantiomers of M1, to investigate the kinetics of the reversible biotransformation of pentoxifylline to (R)- and (S)-M1 in hemolysed erythrocyte suspension, and to quantify the formation of the enantiomers of M1 (as well as M4 and M5) after intravenous and oral administration of pentoxifylline to human volunteers. (R)- and (S)-M1 could be separated preparatively on a cellobiohydrolase column, while determination in blood or plasma was by HPLC after chiral derivatization with diacetyl-L-tartaric acid anhydride. The metabolism of pentoxifylline to (R)-M1 in suspensions of hemolysed erythrocytes followed simple Michaelis-Menten kinetics (K(m) = 11 mM), while that to (S)-M1 was best described by a two-enzyme model (K(m) = 1.1 and 132 mM). Studies with inhibitors indicated that the enzymes were of the carbonyl reductase type. At a therapeutic blood concentration of pentoxifylline, the calculated rate of formation of (S)-M1 is 15 times higher than that of the (R)-enantiomer. Back-conversion of M1 to pentoxifylline was 3-4 times faster for the (S)- than for the (R)-enantiomer. In vivo, the R:S plasma concentration ratio of M1 ranged from 0.010-0.025 after intravenous infusion of 300 or 600 mg of pentoxifylline, and from 0.019-0.037 after oral administration of 600 mg. The biotransformation of pentoxifylline to M1 was thus highly stereoselective in favor of the (S)-enantiomer both in vitro and in vivo.     

Role of high-density lipoprotein in transport of circulating bilirubin in rats

The analbuminemic rat strain established by Nagase et al. (Nagase, S., Shimamune, K., and Shumiya, S. (1979) Science 205, 590-591) exhibits hereditary deficiency in albumin biosynthesis. Serum bilirubin concentration is rather lower in homozygous (aa) rats (0.009 +/- 0.002 mg/dl) as compared with heterozygous (Aa) rats (0.047 +/- 0.009 mg/dl) or wild-type Sprague-Dawley (AA) rats (0.034 +/- 0.006 mg/dl) as evidenced by high pressure liquid chromatography analysis of bilirubin. After intravenous administration of various amounts of [heme-3H]hemoglobin in rats, [3H]bilirubin derived from [3H]heme of hemoglobin in vivo is more efficiently excreted into bile in aa rats than in Aa or AA rats. [3H]Bilirubin is exclusively bound with high-density lipoprotein (HDL) in aa rats, and a significant amount of [3H]bilirubin is shown to bind with HDL in Aa or AA rats in vivo. Scatchard plots revealed that [3H]bilirubin is bound with HDL in three binding modes depending on the molar ratio of [3H]bilirubin to HDL: Kd = 0.8 X 10(-7) M (molar ratio, 0.02-0.06), Kd = 1.6 X 10(-6) M (molar ratio, 0.06-0.41), and Kd = 1.2 X 10(-4) M (molar ratio, 0.79-9.02). Even under extreme conditions of excess hemoglobin administration, the molar ratio remains under 0.041; and thus, expected the Kd value would remain around 0.8 X 10(-7) M. Binding of [3H]bilirubin to rat serum albumin revealed two distinct binding modes depending on the molar ratio of [3H]bilirubin to rat serum albumin: Kd = 3.6 X 10(-7) M (molar ratio, 0.03-0.21), and Kd = 5.0 X 10(-6) M (molar ratio, 0.21-2.46). Under physiological conditions in Aa or AA rats, the former mode would be more reliable than the latter. Thus, HDL could bind with approximately 4.5 times higher affinity than rat serum albumin in Aa or AA rats under physiological conditions in vivo.     

Role of kinins in chronic heart failure and in the therapeutic effect of ACE inhibitors in kininogen-deficient rats

Using Brown Norway Katholiek (BNK) rats, which are deficient in kininogen (kinin precursor) due to a mutation in the kininogen gene, we examined the role of endogenous kinins in 1) normal cardiac function; 2) myocardial infarction (MI) caused by coronary artery ligation; 3) cardiac remodeling in the development of heart failure (HF) after MI; and 4) the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEI) on HF after MI. Two months after MI, rats were randomly treated with vehicle or the ACEI ramipril for 2 mo. Brown Norway rats (BN), which have normal kininogen, were used as controls. Left ventricular (LV) end-diastolic volume (EDV), end-systolic volume (ESV), end-diastolic pressure (EDP), and ejection fraction (EF) as well as myocardial infarct size (IS), interstitial collagen fraction (ICF), cardiomyocyte cross-sectional area (MCA), and oxygen diffusion distance (ODD) were measured. We found that 1) cardiac hemodynamics, function, and histology were the same in sham-ligated BN and BNK rats; 2) IS was similar in BN and BNK; 3) in rats with HF treated with vehicle, the decrease in LVEF and the increase in LVEDV, LVESV, LVEDP, ICF, MCA, and ODD did not differ between BNK and BN; and 4) ACEI increased EF, decreased LVEDV and LVESV, and improved cardiac remodeling in BN-HF rats, and these effects were partially blocked by the bradykinin B(2) receptor antagonist icatibant (HOE-140). In BNK-HF rats, ACEI failed to produce these beneficial cardiac effects. We concluded that in rats, lack of kinins does not influence regulation of normal cardiac function, myocardial infarct size, or development of HF; however, kinins appear to play an important role in the cardioprotective effect of ACEI, since 1) this effect was significantly diminished in kininogen-deficient rats and 2) it was blocked by a B(2) kinin receptor antagonist in BN rats.     

The role of zoos in the rehabilitation of animals in the circus

In 1998, the government of India enforced a ban on performance/exhibition of 5 species of nonhuman animals: (a) lions, (b) tigers, (c) leopards, (d) bears, and (e) monkeys. The Ministry of Environment and Forests gave the responsibility to the Central Zoo Authority (CZA) for rehabilitation of these animals. Between 1999 and 2001, the CZA created rescue centers for rehabilitation of lions and tigers in the off-display areas of 5 zoos: (a) Bangalore, (b) Chennai, (c) Vishakhapatnam, (d) Tirupathi, and (e) Jaipur. Today, the CZA has rehabilitated 314 lions and tigers from circuses. The CZA has been meeting the expenses toward maintenance (feeding of and providing health care for) of the animals, outsourcing of staff, and maintaining enclosures. This article focuses on the mammoth work that the selected zoos had to carry out in rehabilitating the lions and tigers from the circuses.     

Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo

The ADP-ribosyl moiety of NAD+ is consumed in reactions catalyzed by three classes of enzymes: poly(ADP-ribose) polymerase, protein mono(ADP-ribosyl)transferases, and NAD+ glycohydrolases. In this study, we have evaluated the selectivity of compounds originally identified as inhibitors of poly(ADP-ribose) polymerase on members of the three classes of enzymes. The 50% inhibitory concentration (IC50) of more than 20 compounds was determined in vitro for both poly(ADP-ribose) polymerase and mono(ADP-ribosyl)transferase A in an assay containing 300 microM NAD+. Of the compounds tested, benzamide was the most potent inhibitor of poly(ADP-ribose) polymerase with an IC50 of 3.3 microM. The IC50 for benzamide for mono(ADP-ribosyl)transferase A was 4.1 mM, and similar values were observed for four additional cellular mono(ADP-ribosyl)transferases. The IC50 for NAD+ glycohydrolase for benzamide was approximately 40 mM. For seven of the best inhibitors, inhibition of poly(ADP-ribose) polymerase in intact C3H1OT1/2 cells was studied as a function of the inhibitor concentration of the culture medium, and the concentration for 50% inhibition (culture medium IC50) was determined. Culture medium IC50 values for benzamide and its derivatives were very similar to in vitro IC50 values. For other inhibitors, such as nicotinamide, 5-methyl-nicotinamide, and 5-bromodeoxyuridine, culture medium IC50 values were 3-5-fold higher than in vitro IC50 values. These results suggest that micromolar levels of the benzamides in the culture medium should allow selective inhibition of poly(ADP-ribose) metabolism in intact cells. Furthermore, comparative quantitative inhibition studies should prove useful for assigning the biological effects of these inhibitors as an effect on either poly(ADP-ribose) or mono(ADP-ribose) metabolism.     

Regulation of glucose production in rainbow trout: role of epinephrine in vivo and in isolated hepatocytes

The rate of hepatic glucose production (R(a) glucose) of rainbow trout (Oncorhynchus mykiss) was measured in vivo by continuous infusion of [6-(3)H]glucose and in vitro on isolated hepatocytes to examine the role of epinephrine (Epi) in its regulation. By elevating Epi concentration and/or blocking beta-adrenoreceptors with propranolol (Prop), our goals were to investigate the mechanism for Epi-induced hyperglycemia to determine the possible role played by basal Epi concentration in maintaining resting R(a) glucose and to assess indirect effects of Epi in the intact animal. In vivo infusion of Epi caused hyperglycemia (3.75 +/- 0.16 to 8.75 +/- 0.54 mM) and a twofold increase in R(a) glucose (6.57 +/- 0.79 to 13.30 +/- 1.78 micromol. kg(-1). min(-1), n = 7), whereas Prop infusion decreased R(a) from 7.65 +/- 0.92 to 4.10 +/- 0.56 micromol. kg(-1). min(-1) (n = 10). Isolated hepatocytes increased glucose production when treated with Epi, and this response was abolished in the presence of Prop. We conclude that Epi-induced trout hyperglycemia is entirely caused by an increase in R(a) glucose, because the decrease in the rate of glucose disappearance normally seen in mammals does not occur in trout. Basal circulating levels of Epi are involved in maintaining resting R(a) glucose. Epi stimulates in vitro glucose production in a dose-dependent manner, and its effects are mainly mediated by beta-adrenoreceptors. Isolated trout hepatocytes produce glucose at one-half the basal rate measured in vivo, even when diet, temperature, and body size are standardized, and basal circulating Epi is responsible for part of this discrepancy. The relative increase in R(a) glucose after Epi stimulation is similar in vivo and in vitro, suggesting that indirect in vivo effects of Epi, such as changes in hepatic blood flow or in other circulating hormones, do not play an important role in the regulation of glucose production in trout.     

Role of Rho-kinase in reexpansion pulmonary edema in rabbits

Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with (125)I-labeled albumin. After intratracheal instillation of (125)I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of (125)I-albumin in plasma/total amount of instilled (125)I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H(2)O(2)-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema.     

Effect of NADPH-oxidase inhibitors in the experimental model of zymosan-induced shock in mice

The aim of this study was to investigate the effects of NADPH-oxidase inhibitors, in a mouse model of zymosan. Zymosan-induced shock was induced in mice by administration of zymosan (500 mg/kg, i.p.). The pharmacological treatment was the administration of apocynin (5 mg/kg 10% DMSO i.p.) and diphenylene iodonium chloride (DPI) (1 mg/kg i.v.) 1 h and 6 h after zymosan administration. MOF and systemic inflammation in mice was assessed 18 h after administration of zymosan. NADPH-oxidase inhibitors caused a significant reduction of the (1) peritoneal exudate formation, (2) neutrophil infiltration, (3) multiple organ dysfunction syndrome, (4) nitrotyrosine, (5) poly (ADP-ribose) (PAR), (6) cytokine formation, (7) adhesion molecule expression, (8) nuclear factor (NF-κB) expression and (9) apoptosis induced by zymosan. Moreover, NADPH-oxidase inhibitors treatment significantly reduced the systemic toxicity, the loss in body weight and the mortality caused by zymosan. This study has shown that NADPH-oxidase inhibitors attenuate the degree of zymosan-induced non-septic shock in mice.     

Changes in vascular perfusion of the endometrium in association with changes in location of the embryonic vesicle in mares

The equine embryonic vesicle is mobile on Days 12-14 (Day 0 = ovulation), when it is approximately 9-15 mm in diameter. Movement from one uterine horn to another occurs, on average, approximately 0.5 times per hour. Mobility ceases (fixation) on Days 15-17. Transrectal color Doppler ultrasonography was used to study the relationship of embryo mobility (experiment 1) and fixation (experiment 2) to endometrial vascular perfusion. In experiment 1, mares were bred and examined daily from Day 1 to Day 16 and were assigned, retrospectively, to a group in which an embryo was detected (pregnant mares; n = 16) or not detected (n = 8) by Day 12. Endometrial vascularity (scored 1-4, for none to maximal, respectively) did not differ on Days 1-8 between groups or between the sides with and without the corpus luteum. Endometrial vascularity scores were higher (P < 0.05) on Days 12-16 in both horns of pregnant mares compared to mares with no embryo. In pregnant mares, the scores increased (P < 0.05) between Day 10 and Day 12 in the horn with the embryo and were higher (P < 0.05) than scores in the opposite horn on Days 12-15. In experiment 2, 14 pregnant mares were examined from Day 13 to 6 days after fixation. Endometrial vascularity scores and number of colored pixels per cross-section of endometrium were greater (P < 0.05) in the endometrium surrounding the fixed vesicle than in the middle portion of the horn of fixation. Results supported the hypothesis that transient changes in endometrial vascular perfusion accompany the embryonic vesicle as the vesicle changes location during embryo mobility.     

A comparison of in vitro and in vivo stability in mice of two morpholino duplexes differing in chain length

The stability of hybridized duplexes is an important criterion for any radiopharmaceutical application of DNAs or their analogues such as phosphorodiamidate morpholinos (MORFs). OBJECTIVE: The stabilities in vitro and in mice of the duplex between MORF and its complement (cMORF) were investigated for two different chain lengths, a 15-mer MORF compared to the identical MORF but elongated to a 25-mer. METHODS: The hybridization characteristics of the 15-mer MORF with its complementary 15-mer and that of the 25-mer with its complementary 25-mer MORF were measured using surface plasmon resonance (SPR) analysis. For radiolabeling with (99m)Tc, the 15- and 25-mer MORF, both with a primary amine via a 10-member linker on the 3' equivalent end, were conjugated with NHS-MAG(3). The 15- and 25-mer cMORFs were conjugated via their amines to carbodiimidazole treated poly(methyl vinyl ether-alt-maleic acid) (PA) such that about 50 cMORFs were attached to each polymer molecule in both cases (estimated MWs about 300 and 450 kDa, respectively). After hybridization in vitro, both the PA-cMORF15-(99m)Tc-MORF15 and PA-cMORF25-(99m)Tc-MORF25 homoduplexes were evaluated by size exclusion HPLC in saline, after incubation in 37 degrees C serum and in urine obtained 30 min post IV administration to normal mice. Biodistributions were obtained up to 18 h post administration. RESULTS: By SPR, the affinity constants for the homoduplexes were both about 10(9) M(-)(1) with the 25/25 only about 25% higher than the 15/15. However, the affinity constants for the 15/25 and 25/15 heteroduplexes showed a surprisingly 13-fold difference. By HPLC analysis, all duplexes were stable in saline; however, analysis of serum incubates and urine containing PA-cMORF15-(99m)Tc-MORF15 showed an immediate and pronounced low molecular weight peak that was identified by a shift assay to be (99m)Tc-MORF15. The comparable peak in both fluids was much less pronounced in the case of PA-cMORF25-(99m)Tc-MORF25. Whole body radioactivity levels also fell much more rapidly in mice receiving the 15-mer conjugate (65 vs 30% eliminated at 18 h) and biodistribution results showed higher kidney levels for the 15-mer conjugate. Results with the PA-cMORF25-(99m)Tc-MORF15 heteroduplex were more similar to that obtained with the 15-mer homoduplex than the 25-mer homoduplex. CONCLUSION: Despite what is reported to be high hybridization affinities, both the homoduplex and heteroduplexes prepared with (99m)Tc-MORF15 were found to be unstable in serum and in vivo toward dissociation to free (99m)Tc-MORF15. By contrast, homoduplex prepared with (99m)Tc-MORF25 showed higher stability. These differences in hybridization stability may be important considerations in radiopharmaceutical design.     

S-Transnitrosylation of albumin in human plasma and blood in vitro and in vivo in the rat

S-Nitrosoalbumin (SNOALB) is the most abundant physiological circulating nitric oxide (NO) carrier regulating NO-dependent biological actions in humans. The mechanisms of its formation and biological actions are still incompletely understood. Nitrosation by authentic NO and S-transnitrosylation of the single sulfhydryl group located at Cys-34 of human albumin by the physiological S-nitroso compounds S-nitrosocysteine (SNOC) and S-nitrosoglutathione (GSNO) are two possible mechanisms. On a quantitative basis, we investigated by gas chromatography-mass spectrometry the contribution of these two mechanisms to SNOALB formation in human plasma and blood in vitro. GSNO and SNOC (0-100 microM) rapidly and efficiently (recovery=35%) S-transnitrosylated albumin to form SNOALB. NO (100 microM) S-nitrosated albumin to SNOALB at a considerably lower extent (recovery=5%). The putative NO-donating drugs glyceryl trinitrate and sodium nitroprusside (each 100 microM) failed completely in S-nitrosating albumin. Bubbling NO into human plasma and blood resulted in formation of SNOALB that inhibited ADP-induced platelet aggregation. Infusion of GS(15)NO in the rat resulted in formation of S(15)NOALB, [(15)N]nitrate and [(15)N]nitrite. Our results suggest that S-transnitrosylation of albumin by SNOC and GSNO could be a more favored mechanism for the formation of SNOALB in the circulation in vivo than S-nitrosation of albumin by NO itself.     

Hemodynamic consequences of rapid changes in posture in humans.

Tolerance to +G(z) gravitational stress is reduced when +G(z) stress is preceded by exposure to hypogravity (fraction, 0, or negative G(z)). For example, there is an exaggerated fall in eye-level arterial pressure (ELAP) early on during +G(z) stress (head-up tilt; HUT) when this stress is immediately preceded by -G(z) stress (head-down tilt; HDT). The aims of the present study were to characterize the hemodynamic consequences of brief HDT on subsequent HUT and to test the hypothesis that an elevation in leg vascular conductance induced by -G(z) stress contributes to the exaggerated fall in ELAP. Young healthy subjects (n = 3 men and 4 women) were subjected to 30 s of 30 degrees HUT from a horizontal position and to 30 s of 30 degrees HUT when HUT was immediately preceded by 20 s of -15 degrees HDT. Four bouts of HDT-HUT were alternated between five bouts of HUT in a counterbalanced designed to minimize possible time effects of repeated exposure to gravitational stress. One minute was allowed for recovery between tilts. Brief exposure to HDT elicited an exaggerated fall in ELAP during the first seconds of the subsequent HUT (-17.9 +/- 1.4 mmHg) compared with HUT alone (-12.4 +/- 1.2 mmHg, P <0.05) despite a greater rise in stroke volume (Doppler ultrasound) and cardiac output over this brief time period in the HDT-HUT trials compared with the HUT trials (thereafter stroke volume fell under both conditions). The greater fall in ELAP was associated with an exaggerated increase in leg blood flow (femoral artery Doppler ultrasound) and was therefore largely (70%) attributable to an exaggerated rise in estimated leg vascular conductance, confirming our hypotheses. Thus brief exposure to -G(z) stress leads to an exaggerated fall in ELAP during subsequent HUT, owing to an exaggerated increase in estimated leg vascular conductance.     

Species specificity in the biosynthesis of branched paraffins in leaves

Isobutyrate-1-(14)C and l-isoleucine-U-(14)C fed through the petiole labeled the surface lipids of broccoli leaves, but the incorporation was much less than from straight chain precursors. Not more than one-third of the (14)C incorporated into the surface lipids was found in the C(29) paraffin and derivatives, whereas more than two-thirds of the (14)C from straight chain precursors are usually found in these compounds. The small amount of (14)C incorporated into the paraffin fraction was found in the n-C(29) paraffin rather than branched paraffins showing that the (14)C in the paraffin must have come from degradation products. Radio gas-liquid chromatography of the saturated fatty acids showed that, in addition to the n-C(16) acid which was formed from both branched precursors, isoleucine-U-(14)C gave rise to branched C(15), C(17), and C(19) fatty acids, and isobutyrate-1-(14)C gave rise to branched C(16) and C(18) acids. Thus the reason for the failure of broccoli leaf to incorporate branched precursors into branched paraffins is not the unavailability of branched fatty acids, but the absolute specificity of the system that synthesizes paraffins, probably the elongation-decar-boxylation enzyme complex. Consistent with this view, no labeled branched fatty acids longer than C(19) could be found in the broccoli leaf. The branched fatty acids were also found in the surface lipids indicating that the epidermal layer of cells did have access to branched chains. Thus the paraffin synthesizing enzyme system is specific for straight chains in broccoli, but the fatty acid synthetase is not.     

Sequence dependent conformations of oligomeric DNA's in aqueous solutions and in crystals

In order to determine the sequence dependence of the conformation of deoxynucleotides, Raman spectra have been obtained for the following oligodeoxynucleotides in aqueous salt solutions and in crystals: d(CpG)(I), d(TGCGCGCA)(II), d(CACGCGTG)(III), d(CGTGCACG)(IV), d(CGCATGCG)(V), d(ACGCGCGT)(VI), d(CGCGTACGCG)(VII), d(CGCACGTGCG)(VIII) and d(CGTGCGCACG)(IX), d(GCTATAGC) (X), d(GCATATGC) (XI), d(GGTATACC) (XII) and d(GGATATCC) (XIII). The normal B type conformation is observed for all the oligomer DNA's at low salt (0.1-1.0 M NaCl) concentration in the temperature range of 0-25 degrees C. It was considered possible that all of the first nine oligomers could go into the Z form in aqueous high salt (5.0-6.0 M NaCl) solutions, and under these conditions the last four were considered candidates to go into the A form. The B-type conformation was found to exist in high salt solutions for (I), (IV), (V), (VI), (X), (XI) and (XIII); the Z or partial Z conformation appears in high salt solution for the oligomers, (II), (III), (VII), (VIII) and (IX); an A or partial A conformation appears in high salt solution for (XII). In the crystalline state, (IV), (VIII), (X), and (XI) stay in the B-form and all of the other oligomers adopt the complete Z-form except for (XII) which crystallizes in the A form. In both the crystal and in aqueous solutions, the identification of the conformation genus was made by means of Raman spectroscopy. In the crystal of (I), grown at pH7.0, guanosine is found to be in C3'-endo/syn conformation and cytidine in C2'-endo/anti, which may be taken as the ideal building block of the typical Z conformation. At pH4, (I) crystallizes in a conformation similar to the B genus. A study of the thermally induced B to Z transition has been carried out for (II) and (III). Based on the analysis of Raman spectra of the alternating pyrimidine-purine oligomers which might be expected to go into the Z form, the tendency for these oligonucleotides to adopt the Z form can be ranked as: d(CGCGCGCG) greater than (II) greater than (III) greater than (V) approximately (VI) greater than (IV) for octamers and (VII) greater than (VIII) greater than (IX) for the decamers. Similarly, those oligomers which might have a tendency to go into the A form could be ranked as (XII) greater than (XIII) approximately (X) greater than (XI). These data should provide help in formulating rules for predicting the sequence dependence of the B to A and B to Z transitions. Some possible rules are explored, but precautions should be taken.     

Erythrocyte-associated transients in PO2 revealed in capillaries of rat mesentery

Mathematical models have predicted the existence of Po(2) gradients between erythrocytes in capillaries in the usual case where plasma contributes substantial resistance to oxygen diffusion. According to theoretical predictions, these gradients could be detected as rapid Po(2) fluctuations (erythrocyte-associated transients, EATs) along the capillary. However, verification of a model and correct choice of its parameters can be made only on the basis of direct experimental measurements. We used phosphorescence quenching microscopy to measure Po(2) in 52 capillaries of rat mesentery to obtain plasma Po(2) values 100 times/s at a given point along a capillary. A 532-nm laser generated 10-micros pulses of light, concentrated by a x100 objective, onto a spot 0.9 microm in diameter. The presence of erythrocytes in the excitation region was detected on the basis of phosphorescence amplitude (PA), proportional to the amount of plasma encountered by the laser beam, and on the basis of the intensity of transmitted laser light (LT), detected by a photodiode placed under the capillary. The data revealed correlated waveforms in PA, LT, and Po(2) in capillaries. The magnitude of the Po(2) gradients between erythrocytes and plasma was correlated with average capillary Po(2). EATs in Po(2) were more readily detected in capillaries with relatively low oxygenation. The correlation coefficients between PA and Po(2) for the half of the capillaries (n = 26) below the median Po(2) (mean Po(2) = 17 mmHg; R = -0.72) was higher than that for the other half (mean Po(2) = 39 mmHg; R = -0.38). These results support the theoretical predictions of EATs and plasma Po(2) gradients in capillaries.     

A measure of phase ambiguity in pairs of SNPs in the presence of linkage disequilibrium

The extent of haplotype ambiguity in a string of single-nucleotide polymorphisms (SNPs) was quantified by Hodge et al. [Nat Genet 1999;21:360]. In their measure, the level of ambiguity increases with increasing numbers of loci and as loci become more polymorphic. That work assumed linkage equilibrium (LE). However, linkage disequilibrium (LD) provides additional information about the haplotypes at a site, thereby diluting the level of ambiguity. The ambiguity vanishes altogether when LD reaches its maximum value. Here, we introduce the ambiguity measure, Phi, to allow for LD (between pairs of SNPs). We derive the formula Phi = 4x(2)x(3) for ambiguity in individuals, where x(1), x(2), x(3) and x(4) are the probabilities of the A(1)A(2), A(1)B(2), B(1)A(2) and B(1)B(2) haplotypes, respectively, and w.l.o.g. x(1)x(4) > or = x(2)x(3). Alternatively, Phi can be expressed in terms of the allele frequencies and the LD parameter delta. We also extend the formula to triads of two parents plus one child. We estimate our measure Phi for relevant SNPs in the published lipoprotein lipase (LPL) gene dataset [Clark et al., Am J Hum Genet 1998;63:595; Nickerson et al., Nat Genet 1998;19:233], obtaining values ranging from a low of 0 to a high of 0.11 among adjacent pairs of sites. In genome-wide LD studies to map common disease genes, a dense map of SNPs may be utilized to detect association between a marker and disease. Therefore, the measurement of ambiguity can potentially help investigators to determine a more efficient map, designed to minimize ambiguity and subsequent information loss.     

Use of Poisons in Determination of Microbial Manganese Binding Rates in Seawater

A method was developed to determine whether microorganisms mediate the precipitation of manganese(II) in the marine environment. Radioactive ⁵⁴Mn(II) was used as a tracer to measure the precipitation (binding and oxidation) of Mn(II) [i.e., the ⁵⁴Mn(II) trapped on 0.2-μm membrane filters] in the presence and absence of biological poisons. A variety of antibiotics, fixatives, and metabolic inhibitors were tested in laboratory control experiments to select poisons that did not interfere in the chemistry of manganese. The poisons were deemed suitable if (i) they did not complex Mn(II) more strongly than the ion-exchange resin Chelex 100, (ii) they did not interfere in the adsorption of ⁵⁴Mn(II) onto synthetic δMnO₂ (manganate), (iii) they did not cause desorption of ⁵⁴Mn(II) which had been preadsorbed onto synthetic manganate, and (iv) they did not solubilize synthetic ⁵⁴manganate. In addition, several known chelators, reducing agents, and buffers normally added to microbiological growth media or used in biochemical assays were tested. Most additions interfered to some extent with manganese chemistry. However, at least one inhibitor, sodium azide, or a mixture of sodium azide, penicillin, and tetracycline was shown to be appropriate for use in field studies of ⁵⁴Mn(II) binding. Formaldehyde could also be used in short incubations (1 to 3 h) but was not suitable for longer time course studies. The method was applied to studies of Mn(II) precipitation in Saanich Inlet, British Columbia, Canada. Bacteria were shown to significantly enhance the rate of Mn(II) removal from solution in the manganese-rich particulate layer which occurs just above the oxygen-hydrogen sulfide interface in the water column.