Quantitative amino acid compositions at the picomole level using fluorescence scanning

Simple techniques are described for quantitating dansyl amino acids by direct fluoresence scanning and by photo-copying and densitometry. This technique is accurate between 1.0 to 10 × 10^?12 moles with a lower limit of detection around 1 × 10^?14 moles. It is possible to obtain amino acid compositions on very small quantities of peptides and it seems likely that this method will be useful for automatic peptide sequenators which use subtractive dansyl-Edman degradation as a detection procedure.     

Some Operational Characteristics of Glycine Release in Rat Retina: The Role of Reverse Mode Operation of Glycine Transporter Type-1 (GlyT-1) in Ischemic Conditions

Rat posterior eyecups containing the retina were prepared, loaded with [³H]glycine and superfused in order to determine its release originated from glycinergic amacrine cells and/or glial cells. Deprivation of oxygen and glucose from the Krebs-bicarbonate buffer used for superfusion evoked a marked increase of [³H]glycine release, an effect that was found to be external Ca²⁺-independent. Whereas oxygen and glucose deprivation increased [³H]glycine release, its uptake was reduced suggesting that energy deficiency shifts glycine transporter type-1 operation from normal to reverse mode. The increased release of [³H]glycine evoked by oxygen and glucose deprivation was suspended by addition of the non-competitive glycine transporter type-1 inhibitor NFPS and the competitive inhibitor ACPPB further suggesting the involvement of this transporter in the mediation of [³H]glycine release. Oxygen and glucose deprivation also evoked [³H]glutamate release from rat retina and the concomitantly occurring release of the NMDA receptor agonist glutamate and the coagonist glycine makes NMDA receptor pathological overstimulation possible in hypoxic conditions. [³H]Glutamate release was suspended by addition of the excitatory amino acid transporter inhibitor TBOA. Sarcosine, a substrate inhibitor of glycine transporter type-1, also increased [³H]glycine release probably by heteroexchange shifting transporter operation into reverse mode. This effect of sarcosine was also external Ca²⁺-independent and could be suspended by NFPS. Energy deficiency in retina induced by ouabain, an inhibitor of the Na⁺–K⁺-dependent ATPase, and by rotenone, a mitochondrial complex I inhibitor added with the glycolytic inhibitor 2-deoxy-d-glucose, led to increase of retinal [³H]glycine efflux. These effects of ouabain and rotenone/2-deoxy-d-glucose could also be blocked by NFPS pointed to the preferential reverse mode operation of glycine transporter type-1 as a consequence of impaired cellular energy homeostasis. Immunohistochemical studies revealed that glycine transporter type-1, of which reverse mode operation assures [³H]glycine release, is expressed in amacrine cells in the inner nuclear and plexiform layers of the retina and also in Müller macroglia cells. We conclude that disruption of the balanced normal/reverse mode operation of glycine transporter type-1 is likely a significant factor contributing to neurotoxic processes of the retina. The possibility to inhibit glycine transporter type-1 mediated glycine efflux by drugs more potently than glycine uptake might offer some therapeutic potential for the treatment of various neurodegenerative disorders of the retina.     

High level of aspartic acid-bond isomerization during the synthesis of an N-linked tau glycopeptide.

An increased degree of utilization of the potential N-glycosylation site in the fourth repeat unit of the human tau protein may be involved in the inability of tau to bind to the corresponding tubulin sequence(s) and in the subsequent development of the paired helical filaments of Alzheimer's disease. To model these processes, we synthesized the octadecapeptide spanning this region without sugar, and with the addition of an N-acetyl-glucosamine moiety. The carbohydrate-protected, glycosylated asparagine was incorporated as a building block during conventional Fmoc-solid phase peptide synthesis. While the crude non-glycosylated analog was obtained as a single peptide, two peptides with the identical, expected masses, in approximately equal amounts, were detected after the cleavage of the peracetylated glycopeptide. Surprisingly, the two glycopeptides switched positions on the reversed-phase high performance liquid chromatogram after removal of the sugar-protecting acetyl groups. Nuclear magnetic resonance spectroscopy and peptide sequencing identified the more hydrophobic deprotected peak as the target peptide, and the more hydrophilic deprotected peak as a peptide analog in which the aspartic acid-bond just preceding the glycosylated asparagine residue was isomerized resulting in the formation of a beta-peptide. The anomalous chromatographic behavior of the acetylated beta-isomer could be explained on the basis of the generation of an extended hydrophobic surface which is not present in any of the other three glycopeptide variants. Repetition of the syntheses, with altered conditions and reagents, revealed reproducibly high levels of aspartic acid-bond isomerization of the glycopeptide as well as lack of isomerization for the non-glycosylated parent analog. If similar increased aspartic acid-bond isomerization occurs in vivo, a protein modification well known to take place for both the amyloid deposits and the neurofibrillary tangles in Alzheimer's disease, this process may explain the aggregation of glycosylated tau into the paired helical filaments in the affected brains.     

Inhibition of progesterone secretion and decrease of ovarian blood flow by isoxazole in pregnant rats

Pregnant rats were treated on day 11 of pregnancy with 0.62, 1.25, 2.5 or 5.0 mg of isoxazole per 100 g of body weight (bw) (Experiment I) or with 2.5 mg of isoxazole per 100 g of bw (Experiment II). In Experiment I animals were anaesthetized with pentobarbital at 48 hr. In experiment II animals were similarly anaesthetized at 0, 12, 24, or 48 hr following administration of isoxazole. Blood was collected separately from the ovary or from the uterus via a thin cannula which was inserted into the utero-ovarian vein. Blood was also collected from the vena cava inferior. The concentrations of progesterone (P), 17 beta-oestradiol (E2), and corticosterone were measured in blood samples by RIA. All of the animals exhibited reduced rates of ovarian venous blood flow and rates of P secretion when pregnancy was terminated by isoxazole (2.5 mg/100 g bw or greater). The concentration of P was found to be correspondingly reduced by 90% in ovarian venous blood and by 50% in peripheral blood, relative to the controls. After isoxazole had been administered, the rate of P secretion and also the concentration of P in peripheral blood continued to decrease rapidly and to remain at low levels for at least 48 hr. By contrast, the concentration of corticosterone in peripheral blood decreased during the first 12 hr but then it returned to the control levels by 48 hr. The ovarian secretion rate of E2 and the concentration of E2 in peripheral blood remained unchanged during the same 48 hr period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multilevel studies on the two phenological forms of Large Blue (Maculinea arion) (Lepidoptera: Lycaenidae)

The main goal of our research was to study comprehensively the differences between the two phenological forms of the socially parasitic and globally threatened Large Blue (Maculinea arion) in the Carpathian Basin using four character sets (mitochondrial sequences, allozymes, male genitalia and wing morphometrics). Comparative analyses of distance matrices, phylogenetic trees and ordination patterns have been applied. The genetic and morphometric patterns revealed by our studies were discordant. While we experienced a significant differentiation between the ‘spring’ and ‘summer type’ of M. arion in both wing and genital traits, the two phenological forms did not show any genetic differentiation on two mitochondrial loci and in allozymes. At the same time, all individuals were infected by Wolbachia. Although certain wing traits may not represent reliable tracers of phylogeny because of the particular adaptive significance, the wing characteristics involved in our research are probably determined genetically. Additionally, the significant differentiation of male genitalia also indicates incipient prezygotic isolation arising from phenological differentiation between the ‘spring and summer arion’. It is possible that all extant differences between the two forms are attributable to (1) different host‐ant use, (2) incipient speciation, (3) cytoplasmatic incompatibility (CI) by Wolbachia or the combination of these factors. In addition, discordant results indicate that the combined use of different approaches and data sets is strictly necessary to clarify systematic and evolutionary relationships.