Brain 2-phenylethylamine as a major mediator for the central actions of amphetamine and methylphenidate.

2-Phenylethylamine (PEA) was measured in rabbit brain by gas-liquid chromatography. D-Amphetamine sulfate (0.65 mg/Kg) initially reduced brain PEA levels to one-third of its usual content (30 min) and subsequently doubled brain PEA (4 hr). Brain PEA levels were reduced (30 min) and subsequently increased (ten-fold at 4 hr) by D-amphetamine sulfate (13 mg/Kg); tolerance to these two effects was observed in rabbits treated for three days with D-amphetamine. Methylphenidate HCl (30 mg/Kg) but not L-amphetamine sulfate (0.65 mg/Kg and 13 mg/Kg) induced a small, non-significant lowering of brain PEA (30 min) followed by a marked augmentation (4 hr) of brain PEA content. D-Amphetamine (30 min or 4 hr prior) increased the recovery of labeled PEA from the brain of rabbits injected intraventricularly with labeled phenylalanine, and reduced the recovery of labeled PEA after its intraventricular injection, suggesting that D-amphetamine accelerates both the synthesis and the disposition of brain PEA. Pretreatment with α-methyldopa (which depletes PEA and other brain amines) or with α-methyldopa hydrazine (which selectively reduces brain PEA content by inhibiting decarboxylase in peripheral tissues only) markedly reduced the CNS effects of D-amphetamine (behavioral stimulation in mice and rabbits, anti-convulsant effect in mice); these decarboxylase inhibitors enhanced the amphetamine-like effects induced by PEA in mice pretreated with a monoamine oxidase inhibitor. The ability of PEA depleters to selectively block the stimulant effects of D-amphetamine, together with the close structural and pharmacological similarities between amphetamine and PEA, and marked influence of amphetamine administration upon PEA brain levels, synthesis and metabolism, suggest to us that many of the central actions of amphetamine may be mediated by endogenous PEA.     

Molecular evolutionary dynamics of cytochrome b in strepsirrhine primates: the phylogenetic significance of third-position transversions

DNA sequences of the complete cytochrome b gene are shown to contain robust phylogenetic signal for the strepsirrhine primates (i.e., lemurs and lorises). The phylogeny derived from these data conforms to other molecular studies of strepsirrhine relationships despite the fact that uncorrected nucleotide distances are high for nearly all intrastrepsirrhine comparisons, with most in the 15%-20% range. Cytochrome b sequences support the hypothesis that Malagasy lemuriforms and Afro-Asian lorisiforms each comprise clades that share a sister- group relationship. A study (Adkins and Honeycutt 1994) of the cytochrome c oxidase subunit II (COII) gene placed one Malagasy primate (Daubentonia) at the base of the strepsirrhine clade, thereby suggesting a diphyletic Lemuriformes. The reanalysis of COII third- position transversions, either alone or in combination with cytochrome b third-position transversions, however, yields a tree that is congruent with phylogenetic hypotheses derived from cytochrome b and other genetic data sets.      

Further evidence for a role of 2-phenylethylamine in the mode of action of delta 9-tetrahydrocannabinol

In rabbits, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) increased the recovery of labeled 2-phenylethylamine (PEA) from brain following its intraventricular administration. Δ⁹-THC also enhanced the excitatory effect of iontophoretic PEA on cortical unit potentials. Although Δ⁹-THC induced sedation in mice, the subsequent injection of reserpine induced transient excitement. Low doses of PEA, which do not significantly alter the behavior of mice, induced marked excitement in mice pretreated with Δ⁹-THC. In mice treated with pargyline, Δ⁹-THC induced excitement (instead of sedation); this excitement was increased by PEA and reduced by phenylethanolamine. These results suggest that Δ⁹-THC inhibits the disposition of PEA. Since endogenous PEA may be one of the adrenergic ergotropic modulators, it may play a role in the euphoriant effect of marihuana.     

The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants

The photosynthetic gene rbcL has been lost or dramatically altered in some lineages of nonphotosynthetic parasitic plants, but the dynamics of these events following loss of photosynthesis and whether rbcL has sustained functionally significant changes in photosynthetic parasitic plants are unknown. To assess the changes to rbcL associated with the loss of functional constraints for photosynthesis, nucleotide sequences from nonparasitic and parasitic plants of Scrophulariales were used for phylogeny reconstruction and character analysis. Plants in this group display a broad range of parasitic abilities, from photosynthetic ("hemiparasites") to nonphotosynthetic ("holoparasites"). With the exception of Conopholis (Orobanchaceae), the rbcL locus is present in all parasitic plants of Scrophulariales examined. Several holoparasitic genera included in this study, including Boschniakia, Epifagus, Orobanche, and Hyobanche, have rbcL pseudogenes. However, the holoparasites Alectra orobanchoides, Harveya capensis, Harveya purpurea, Lathraea clandestina, Orobanche corymbosa, O. fasciculata, and Striga gesnerioides have intact open reading frames (ORFs) for the rbcL gene. Phylogenetic hypotheses based on rbcL are largely in agreement with those based on sequences of the nonphotosynthetic genes rps2 and matK and show a single origin of parasitism, and loss of photosynthesis and pseudogene formation have been independently derived several times in Scrophulariales. The mutations in rbcL in nonparasitic and hemiparasitic plants would result in largely conservative amino acid substitutions, supporting the hypothesis that functional proteins can experience only a limited range of changes, even in minimally photosynthetic plants. In contrast, ORFs in some holoparasites had many previously unobserved missense substitutions at functionally important amino acid residues, suggesting that rbcL genes in these plants have evolved under relaxed or altered functional constraints.      

Alternative splicing and protein function

Background  

Alternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data.     

Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components

The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues.     

Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8- fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400- fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.