Applying the Taguchi robust design to the optimization of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers' yeast

This study examined the characteristics and operational parameters of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast in order to produce S-4-chloro-3-hydroxybutyric acid ethyl ester. Eight operational variables were also optimized using the Taguchi method with consideration of the freshness of yeast cells as a noise factor. An L₁₈ orthogonal array was used to design the experiments. The reaction yield and the product's optical purity were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Additionally, the signal-to-noise (SN) ratio was used to estimate the variability in product quality. Optimization was undertaken not only to yield the best performance, but also to minimize the variation in quality. The confirmation experiments indicated that the reaction performance and the robustness of the product quality under the optimized conditions were higher than those obtained in other experiments in this study. Our results further demonstrate that the product's optical purity could be increased to >95% by adjusting the operational level of the main factors.     

Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda-carrageenan-induced inflammatory pain

In this study, we examined the effect of lambda-carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% lambda-carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed lambda-carrageenan significantly increased brain uptake of [(14)C]sucrose at 1, 3, 6, and 48 h (139 +/- 9%, 166 +/- 19%, 138 +/- 13%, and 146 +/- 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that lambda-carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1-6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.     

Nucleotide variation in the triosephosphate isomerase (Tpi) locus of Drosophila melanogaster and Drosophila simulans

DNA sequence variation in a 1.1-kb region including the coding portion of the Tpi locus was examined in 25 homozygous third-chromosome lines of Drosophila melanogaster, nine lines of Drosophila simulans, and one line of Drosophila yakuba. Our data show that the widespread allozyme polymorphism observed in cosmopolitan D. melanogaster is due to a glutamic acid substitution occurring in a phylogenetically conserved lysine that has been identified as part of the "hinged-lid" active site of the enzyme. This observation suggests that the replacement polymorphism may have important functional consequences. One replacement polymorphism was also observed in D. simulans, although its functional relevance is more difficult to assess, since it affects a site that is not strongly conserved. This amino acid change in D. simulans is associated with a single lineage possessing seven unique silent substitutions, which may be indicative of balancing selection or population subdivision. The absence of fixed amino acid differences between D. melanogaster and D. simulans and only a single difference with D. yakuba suggests that triose phosphate isomerase is under strong functional constraint. Silent variation is slightly higher for D. melanogaster than for D. simulans. Finally, we outline the general lack of evidence for old balanced polymorphisms at allozyme loci in D. melanogaster.      

Melatonin facilitates synchronization of sparrow circadian rhythms to light

We recorded circadian locomotor activity rhythms of house sparrows (Passer domesticus) exposed to low-amplitude light-dark cycles (2∶1 lux) with periods of 22.5 or 24.5 h. Under these conditions the circadian rhythms of the majority of the birds were not synchronized by the light cycle but either free-ran or showed relative coordination. However, when melatonin was administered continuously via subcutaneous silastic implants the rhythms became synchronized. It is proposed that melatonin facilitates synchronization either by weakening the circadian oscillatory system thereby increasing its range of entrainment, or by enhancing circadian sensitivity to the light Zeitgeber. In general, the results suggest that melatonin, besides its well-known phasic effects on the circadian system also has important tonic effects modifying the ease with which circadian systems can be entrained.     

Demonstration of an analogue to human pregnancy zone protein in the African green monkey.

A serum protein in the grivet (Cercopithecus aethiops), immunologically cross-reacting with human pregnancy zone protein (PZP), is found in males as well as in nonpregnant females. The electrophoretic mobility, antigenic cross-reactivity, molecular weight, and carbohydrate structure appear analogous to PZP; however, contrary to the situation in man, exogenous estrogens cause a fall in serum concentration of the protein.     

Intracerebral and intrathecal infusion of the TGF-beta 2-specific antisense phosphorothioate oligonucleotide AP 12009 in rabbits and primates: toxicology and safety

Here, we provide first evidence that long-term continuous infusion of highly purified antisense phosphorothioate oligodeoxynucleotides (S-ODN) into brain parenchyma is well tolerated and thus highly suitable for in vivo application. AP 12009 is an S-ODN for the therapy of malignant glioma. It is directed against human transforming growth factor-beta (TGF-beta2) mRNA. In the clinical setting, AP 12009 is administered intratumorally by continuous infusion directly into the brain tumor. In view of this clinical application, the focus of our data is on local toxicology studies in rabbits and monkeys to evaluate the safety of AP 12009. AP 12009 was administered either by intrathecal bolus injection into the subarachnoidal space of the lumbar region of both cynomolgus monkeys and rabbits or by continuous intraparenchymatous infusion directly into the brain tissue of rabbits. Intrathecal bolus administration of 0.1 ml of 500 microM AP 12009 showed neither clinical signs of toxicity nor macroscopically visible or histomorphologic changes. After a 7-day intraparenchymatous continuous infusion of 500 microM AP 12009 at 1 microl/h in rabbits, there was no evidence of toxicity except for local mild to moderate lymphocytic leptomeningoencephalitis. Additionally, AP 12009 showed good tolerability in safety pharmacology as well as in acute toxicity studies and 4-week subchronic toxicity studies in mice, rats, and monkeys. This favorable safety profile proves the suitability of AP 12009 for local administration in brain tumor patients from the point of view of toxicology.     

Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1

The BRCT repeats in BRCA1 are essential for its tumor suppressor activity and interact with phosphorylated protein targets containing the sequence pSer-X-X-Phe, where X indicates any residue. The structure of the tandem BRCA1 BRCT repeats bound to an optimized phosphopeptide reveals that the N-terminal repeat harbors a conserved BRCT phosphoserine-binding pocket, while the interface between the repeats forms a hydrophobic groove that recognizes the phenylalanine. Crystallographic and biochemical data suggest that the structural integrity of both binding sites is essential for peptide recognition. The diminished peptide-binding capacity observed for cancer-associated BRCA1-BRCT variants may explain the enhanced cancer risks associated with these mutations.     

Timing of breeding in variable environments: tropical birds as model systems

Animals need to adjust reproductive decisions to environmental seasonality. In contrast to species from the well-studied temperate zones, little is known for tropical birds about the environmental cues that stimulate reproductive activity and the physiological mechanisms that regulate reproduction. I am investigating the environmental and endocrine mechanisms that underlie the timing of reproduction in spotted antbirds from the near-equatorial Panamanian rainforest and in small ground finches from the equatorial arid Galápagos islands. Spotted antbirds live in a fairly predictable seasonal environment and show regular changes in gonad sizes and some reproductive hormones. Despite the small annual variation in photoperiod close to the equator, these birds can measure slight photoperiodic increases and use it to initiate reproductive activity. Spotted antbirds also respond to seasonal changes in food availability, which allows them to flexibly adjust gonad growth to environmental conditions. Testosterone is involved in regulating song and aggressive behavior in these year-round territorial birds, although it can remain at low plasma levels throughout the year. In contrast, small ground finches exposed to a rather unpredictable climate on Galápagos appear to grow their gonads whenever heavy rains fall and have regressed gonads during other times of the year. The lack of a physiological preparation for the breeding season and their response to short-term cues related to rainfall indicate a striking flexibility in the regulation of breeding in small ground finches. I suggest that tropical birds can serve as model systems to study the physiological adaptations to different environments. Unraveling the neuroendocrine mechanisms behind the flexibility in reproductive timing will clarify whether differences found between temperate and tropical birds represent variations of the same basic mechanism or instead reflect a fundamental divergence in physiological control systems.