Base-catalyzed racemization of 3-O-acyloxazepam

Enantiomers of 3-O-acyloxazepam (oxazepam 3-acetate; OXA) underwent base-catalyzed hydrolysis and racemization. Kinetics of reaction products formed from an OXA enantiomer in buffered and unbuffered alkaline solutions were analyzed by chiral stationary phase high-performance liquid chromatography. Racemization occurred with varying rates in aqueous solutions with pH ranging from 7.5 to 14. Racemization mechanism was studied by the dependence of rates of hydrolysis and racemization on temperature and pH. Mass spectral analysis of racemization products derived from an OXA enantiomer in a deuterated solvent indicated that racemization was accompanied by a proton exchange with the solvent. The results indicated that a base-catalyzed keto-enol tautomerism between the C2-carbonyl group and the C3 carbon was responsible for the observed racemization. © 1994 Wiley-Liss, Inc.

1 This article is a US Goverment work and, as such, is in the public domain in the United States of America.

     

Stereoselective nucleophilic substitution of oxazepam and racemization in acidic methanol and ethanol

Enantiomeric and racemic oxazepam (OX), 3-O-methyloxazepam (MeOX), and 3-O-ethyloxazepam (EtOX) were used to study racemization, heteronucleophilic, and homonucleophilic substitution reactions in anhydrous acidic methanol and ethanol. Kinetics of racemization and nucleophilic substitution reactions in nondeuterated and deuterated solvents were determined by circular dichroism spectropolarimetry, chiral stationary phase high-performance liquid chromatography (HPLC), reversed-phase HPLC, and mass spectrometry. Several reactions occurred when (S)-OX, for example, was dissolved in acidic methanol: (1) (S)-OX itself underwent spontaneous racemization, (2) the 3-hydroxyl group of (S)-OX was stereoselectively substituted by the methoxy group of methanol to form MeOX enriched in (S)-MeOX, (3) the 3-methoxy group of (S)-MeOX was stereoselectively substituted by the methoxy group of methanol to form MeOX enriched in (S)-MeOX, and (4) the 3-methoxy group of (R)-MeOX was stereoselectively substituted by the methoxy group of methanol to form MeOX enriched in (R)-MeOX. Repetitive reactions 3 and 4 eventually resulted in a racemic MeOX. Similar reactions occurred for an enantiomeric OX in acidic ethanol. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Enantioselective hydrolysis of oxazepam 3-acetate by esterases in human and rat liver microsomes and rat brain S9 fraction

Rates of hydrolysis of racemic and enantiomeric oxazepam 3-acetates (OXA) by esterases in human and rat liver microsomes and rat brain S9 fraction were compared. When rac-OXA was the substrate, esterases in human and rat liver microsomes were highly enantioselective toward (R)-OXA. In contrast, esterases in rat brain S9 fraction were highly enantioselective toward (S)-OXA. Hydrolysis rates of rac-OXA were highly dependent on the amount of esterases used. At 0.05 mg protein equivalent of esterases and 150 nmol of rac-OXA per ml of incubation mixture, the (R)-OXA was hydrolyzed 3.6-fold and 18.5-fold faster than (S)-OXA by rat and human liver microsomes, respectively. The specific activities (nmol of OXA hydrolyzed/mg microsomal protein/min) of liver microsomes in the hydrolysis of enantiomerically pure (R)-OXA were approximately 120 (rat) and 1,980 (human), and in the hydrolysis of enantiomerically pure (S)-OXA were 4 (rat) and 7 (human), respectively. In the incubation of rac-OXA with rat brain S9 fraction, (S)-OXA was hydrolyzed approximately 6-fold faster than (R)-OXA. Results also indicated an enantiomeric interaction in the hydrolysis of rac-OXA by esterases in rat and human liver microsomes; the presence of (R)-OXA stimulated the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA inhibited the hydrolysis of (R)-OXA. In rat brain S9 fraction, the presence of (R)-OXA inhibited the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA appeared to have stimulated the hydrolysis of (R)-OXA.     

Photoperiod regulation of neuron death in the adult canary

The avian brain undergoes naturally occurring cell death and neuronal replacement in adulthood. Little is known about how neuron survival in adult birds is regulated. However, previous work suggests that this process is open to environmental control. We now report that a reduction in day length from springlike to fall-like conditions can dramatically increase cell death in adult male canaries. Many of the dying cells are projection neurons in the motor pathway controlling song learning and production. Circulating levels of gonadal steroids were not correlated with photoperiod-induced changes in the magnitude of cell death. Our results suggest that neuronal death in adult male canaries is regulated by seasonal changes in photoperiod, and that this occurs independent of chronic changes in gonadal steroid hormone levels. Day length may serve as a predictive environmental cue to time cell death in accordance with seasonal reproduction. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 223–231, 1997

1 This is a US Government work and, as such, is in the public domain in the United States of America.

     

Sense organs on the antennal flagellum of mayflies (Ephemeroptera)

A few very small sensory pegs with the characteristics of chemoreceptors are present in both males and females on the antennal flagellum of the mayflies, Stenacron interpunctatum (Say)

1 Stenacron interpunctatum (Say) is Stenonema interpunctatum (Say) of earlier literature.

and Cloeon sp. They are situated near the proximal end of the flagellum on its ventro-lateral surface and have escaped the attention of earlier investigators.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs: I. Chlorfluazuron,diflubenzuron, and teflubenzuron inhibit incorporation but not uptake of [14C]N-acetyl-D-glucosamine

The purpose of this study was to determine the effects of potent inhibitors of chitin synthesis on an organ culture test system as a basis for determining the mode of action of such compounds. Consequently, we investigated the action of chlorfluazuron (CFA), diflubenzuron (DFB), and teflubenzuron (TFB) on uptake and incorporation into chitin of [¹⁴C]N-acetyl-D-glucosamine ([¹⁴C]GlcNAc) in wing imaginal discs cultured in vitro. Spodoptera frugiperda wing imaginal discs provided a highly responsive test system for studying the inhibition of ecdysteroid-dependent chitin synthesis in a target tissue in vitro. All three inhibitors blocked ecdysteroid-dependent [¹⁴C]GlcNAc incorporation into chitin by the wing imaginal discs. The effectiveness of the inhibitors was not affected by the time of their application, i.e., exposures before, during, or after 20-hydroxyecdysone treatment were equally effective in inhibiting chitin synthesis. Thus, exposure of freshly dissected discs to CFA for periods as short as 15 min inhibited approximately 90% of the chitin synthesis measured 72 h later. In contrast to previous in vivo studies all three inhibitors were similar in their effectiveness in vitro. However, while all three compounds inhibited [¹⁴C]GlcNAc incorporation in a similar dose-dependent manner, only DFB and TFB reduced but did not block uptake of GlcNAc. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Effect of crosslinking on mitochondrial structure and function

Rat liver mitochondria were treated with ethylacetimidate

1 Abbreviations: SDS, sodium dodecylsulfate; DMS, dimethylsuberimidate; EA, ethylacetimidate; MBI, methylbutyrimidate; TMPD, N, N′, N′-tetramethyl-p-phenylenediamine.

and methylbutyrimidate, monofunctional imidates, and with dimethylsuberimidate, a bifunctional imidate, and the effects on structure and function studied. Mitochondria treated with 5 mM dimethylsuberimidate or greater did not respond osmotically when placed in deionized water. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that at concentrations > 5 mM dimethylsuberimidate nearly all mitochondrial polypeptides failed to enter 6% gels, indicating crosslinking of both membrane and soluble proteins. Extensive amidination by ethylacetimidate and methylbutyrimidate had little effect on ascorbate-tetramethylphenylenediamine oxidase while extensive inhibition resulted from dimethylsuberimidate treatment. The possible involvement of molecular motion in electron transport is discussed.     

Identification of pig primordial germ cells by immunocytochemistry and lectin binding

Monoclonal antibodies anti-SSEA-1 and EMA-1, and the lectins DBA and LTA, bound to the surface of large, round cells randomly distributed in the 26-day pig genital ridge. Other antibodies, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, did not react with any cells in the pig genital ridge. SSEA-1-positive cells displayed pseudopods and appeared to migrate from the dorsal mesentery of the hindgut (18-day) to the primordium of the gonad (day 23) and entered the genital ridge by 26 days. The number of SSEA-1-positive cells associated with the dorsal mesentery and genital ridge markedly increased from the 18-day to the 26-day pig embryo. It was concluded that the SSEA-1-positive cells were primordial germ cells (PGCs). Using these markers and alkaline phosphatase histochemistry, pig PGCs derived from the 26-day genital ridge showed no proliferation when grown in STO co-culture in the presence of human LIF, bFGF and SCF. Mol. Reprod. Dev. 46:567–580, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Regulation of neurogenesis by growth factors and neurotransmitters

The generation of neurons and glia in the developing nervous system is likely to be regulated by extrinsic factors, including growth factors and neurotransmitters. Evidence from in vivo and/or in vitro systems indicates that basic fibroblast growth factor, transforming growth factor (TGF)-α, insulin-like growth factor-1, and the monoamine neurotransmitters act to increase proliferation of neural precursors. Conversely, glutamate, γ-aminobutyric acid, and opioid peptides are likely to play a role in down-regulating proliferation in the developing nervous system. Several other factors, including the neuropeptides vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide, as well as the growth factors platelet-derived growth factor, ciliary neurotrophic factor, and members of the TGF-β family, have different effects on proliferation and differentiation depending on the system examined. Expression of many of these factors and their receptors in germinal regions of the central nervous system suggests that they can act directly on precursor populations to control their proliferation. Together, the findings discussed here indicate that proliferation and cell fate determination in the developing brain are regulated extrinsically by complex interactions between a relatively large number of growth factors and neurotransmitters. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 287–306, 1998

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

The evolutionary history of the first three enzymes in pyrimidine biosynthesis

Some metabolic pathways are nearly ubiquitous among organisms: the genes encoding the enzymes for such pathways must therefore be ancient and essential. De novo pyrimidine biosynthesis is an example of one such metabolic pathway. In animals a single protein called CAD

1 Abbreviations: CAD, trifunctional protein catalyzing the first three steps of de novo pyrimidine biosynthesis in higher eukaryotes; CPS, carbamyl phosphate synthetase domain; CPSase, carbamyl phosphate synthetase activity; ATC, aspartate transcarbamylase domain; ATCase, aspartate transcarbamylase activity; DHO, dihydroorotase domain; DHOase, dihydroorotase activity; GLN, glutaminase subdomain or subunit of carbamyl phosphate synthetase, GL Nase, glutaminase activity; SYN, synthetase subdomain or subunit of carbamyl phosphate synthetase; SYNase, synthetase activity.

carries the first three steps of this pathway. The same three enzymes in prokaryotes are associated with separate proteins. The CAD gene appears to have evolved through a process of gene duplication and DNA rearrangement, leading to an in-frame gene fusion encoding a chimeric protein. A driving force for the creation of eukaryotic genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway. The analogous structure in bacteria is the operon. Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated genes.     

Environmental magnetic fields inhibit the antiproliferative action of tamoxifen and melatonin in a human breast cancer cell line

We have previously reported that environmental-level magnetic fields (1.2 μT [12 milligauss], 60 Hz) block the growth inhibition of the hormone melatonin (10⁻⁹ M) on MCF-7 human breast cancer cells in vitro. We now report that the same 1.2 μT, 60 Hz magnetic fields significantly block the growth inhibitory action of pharmacological levels of tamoxifen (10⁻⁷ M). In biophysical studies we have taken advantage of Faraday's Law of Current Induction and tested whether the 1.2 μT magnetic field or the associated induced electric field is responsible for this field effect on melatonin and tamoxifen. We observe that the magnetic field component is associated with the field blocking effect on melatonin and tamoxifen function. To our knowledge the tamoxifen studies represent the first experimental evidence for an environmental-level magnetic field modification of drug interaction with human breast cancer cells. Together, these findings provide support to the theory that environmental-level magnetic fields can act to modify the action of a drug or hormone on regulation of cell proliferation. Melatonin and tamoxifen may act through different biological pathways to down-regulate cell growth, and further studies are required to identify a specific biological site of interaction for the 1.2 μT magnetic field. Bioelectromagnetics 18:555–562, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

The transformation of τ-particles into T4 heads. I. Evidence for the conservative mode of this transformation.

Asynchroneous T4 phage head maturation includes the step of P23 cleavage: P23 of head-related τ-particles is cleaved into P23

1 This paper is part of the thesis of R. K. L. Bijlenga. It is number X of the series: “Studies on the morphopoieses of the head of phage T-even.”

of capsids with a conservative mode of transformation as evidenced by “heavy” labeling in temperature shift-down experiments with mutant 24 (tsL90). Assuming a subunit pool, data indicate in situ cleavage on individual precursor particles. The interpretation becomes less interesting when assuming a compartmentation of the membrane surface; this hypothesis is not ruled out.     

Intracellular calcium signaling by jurkat T-lymphocytes exposed to a 60 hz magnetic field

To explore possible biochemical mechanisms whereby electromagnetic fields of around 0.1 mT might affect immune cells or developing cancer cells, we studied intracellular calcium signaling in the model system Jurkat E6-1 human T-leukemia cells during and following exposure to a 60 Hz magnetic field. Cells were labeled with the intracellular calcium-sensitive fluorescent dye Fluo-3, stimulated with a monoclonal antibody against the cell surface structure CD3 (associated with ligand-stimulated T-cell activation), and analyzed on a FACScan flow-cytometer for increases in intensity of emissions in the range of 515–545 nm. Cells were exposed during or before calcium signal-stimulation to 0.15 mT_rms 60 Hz magnetic field. The total DC magnetic field of 78.2 μT was aligned 17.5° off the vertical axis. Experiments used both cells cultured at optimal conditions at 37 °C and cells grown under suboptimal conditions of 24 °C, lowered external calcium, or lowered anti-CD3 concentration. These experiments demonstrate that intracellular signaling in Jurkat E6-1 was not affected by a 60 Hz magnetic field when culture and calcium signal-stimulation were optimal or suboptimal. These results do not exclude field-induced calcium-related effects further down the calcium signaling pathway, such as on calmodulin or other calcium-sensitive enzymes. Bioelectromagnetics 18:439–445, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Distributions of measurement errors for single-axis magnetic field meters during measurements near appliances

Comparisons are made between the average magnetic flux density as it would be measured with a single-axis coil probe and the flux density at the center of the probe, assuming that the probe is oriented to measure the maximum field at that point. Probability distributions of the differences between the two quantities are calculated assuming a dipole magnetic field and are found to be asymmetric. The distributions are used to estimate the uncertainty for maximum magnetic field measurements at distances that are large compared with the dimensions of the field source. Bioelectromagnetics 18:273–276, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Protein and DNA contents in sperm from an infertile human male possessing protamine defects that vary over time

Sperm from 2 semen samples collected 6 months apart from an infertile male and 3 semen samples collected over an 18-month period from a fertile human male volunteer have been analyzed for their protamine and DNA content. Hup1M and Hup2b antibodies were used to detect the presence of protamines and protamine precursors in western blots of nuclear proteins isolated from pools of sperm. Phosphorus and sulfur contents, which can be used to estimate the nuclear DNA and protamine contents of sperm from fertile males, were measured within individual sperm heads from each semen sample by particle induced x-ray emission (PIXE). The single-cell data reveal no significant differences in the phosphorus and sulfur contents of sperm heads in the three semen samples obtained from the fertile male. For the initial semen sample produced by the infertile male, Western blot data show a normal complement of protamine 1, small amounts of mature protamine 2, and reveal large amounts of anti-protamine 2 reactive proteins with electrophoretic mobilities similar to protamine 2 precursors. Data from PIXE show elevated levels of sulfur within sperm heads compared with sperm from the fertile male. Western blot data exhibit no evidence of protamines or protamine 2 precursors in the second semen sample produced by the infertile male. Data from PIXE suggest that these sperm are highly deficient in sulfur and protamines. These results show that the degree of maturation of sperm cells present in the semen of some infertile males can vary with time. Mol. Reprod. Dev. 50:345–353, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

PBAN regulation of pheromone biosynthesis in female tobacco hornworm moths,Manduca sexta (L.)

Manduca sexta females that were decapitated produced no pheromone during the scotophase following decapitation, indicating that they were free of pheromone biosynthesis activating neuropeptide (PBAN). When deuterated hexadecanoic or (Z)-11-hexadecenoic acid was applied to the sex pheromone glands of decapitated or intact females of the same age, and allowed to incubate in vivo for 24 h, deuterium labeled Δ-11- and Δ-10, 12-unsaturated 16-carbon fatty acids were produced in both types of females. Injection of PBAN into intact or decapitated females 23 h after application of labeled acids had no effect on the production of unsaturated labeled fatty acids. However, deuterium labeled aldehydes were produced only in females that were injected with PBAN. Therefore, in this species, PBAN activates the process by which fatty acyl precursors in the pheromone gland are converted into the pheromonal aldehydes. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Increased expression of heme oxygenase-1 in human retinal pigment epithelial cells by transforming growth factor-β

Antibodies specific for heme oxygenase-1 (HO-1) were produced in rabbits, using the multiple antigen peptide (MAP) technique, and were employed to investigate the ability of transforming growth factor-β1 (TGF-β1) to induce the HO-1 protein in cultured human retinal pigment epithelial (RPE) cells. Western blot analyses showed that the cytokine induced HO-1 in these cells in a time- and dose-dependent manner. TGF-β1 also increased the mRNA for HO-1 in treated cells prior to the increase in HO-1 protein. The induction was effectively blocked by a neutralizing antibody preparation against TGF-β1. When tested under similar conditions, other growth factors such as basic fibroblast growth factor-I, plateletderived growth factor, insulin-like growth factor, transforming growth factor-α, and epidermal growth factor did not show appreciable induction of HO-1. Lipopolysaccharide, tumor necrosis factor-α, and interferon-γ were also not inducers, although TGF-β2 effectively induced HO-1. Heavy metal ions and thiol reagents were also highly potent inducers of HO-1 in human RPE cells. The induction of HO-1 by TGF-β1 was also observed in bovine choroid fibroblasts, but not in HELA, HEL or bovine corneal fibroblasts. Our results demonstrate for the first time that HO-1 can be induced by an important cytokine, TGF-β1, causing an increase in the expression of both HO-1 message and protein in specific neuroepithelial and fibroblast cells. © 1994 wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.