l-[3H]Nitroarginine and l-[3H]Arginine Uptake into Rat Cerebellar Synaptosomes: Kinetics and Pharmacology

Abstract: Characteristics of the transport of the nitric oxide synthase substrate l -arginine and its inhibitor, N ^G-nitro- l -arginine ( l -NOARG), into rat cerebellar synaptosomes were studied. Uptake of both l -arginine and l -NOARG was linear with increasing amount of protein (up to 40 µg) and time of incubation (up to 5 min) at 37°C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of l -NOARG (650 pmol/mg of protein) was three to four times higher than that of l -arginine (170 pmol/mg of protein). l -NOARG uptake showed biphasic kinetics ( K _{m 1} = 0.72 m M , V _{max 1} = 0.98 nmol/min/mg of protein; K _{m 2} = 2.57 m M , V _{max 2} = 16.25 nmol/min/mg of protein). l -Arginine uptake was monophasic with a K _m of 106 µ M and a V _max of 0.33 nmol/min/mg of protein. l -NOARG uptake was selectively inhibited by l -NOARG, N ^G-nitro- l -arginine methyl ester, and branched-chain and aromatic amino acids. l -Alanine and l -serine also inhibited l -NOARG uptake but with less potency. Uptake of l -arginine was selectively inhibited by N ^G-monomethyl- l -arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, l -NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas l -arginine is transported by the basic amino acid carrier system y⁺ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of l -NOARG and l -arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.     

Unilateral incompatibility as a major cause of skewed segregation in the cross between Lycopersicon esculentum and L. pennellii

Skewed segregations are frequent events in segregating populations derived from different interspecific crosses in tomato. To determine a basis for skewed segregations in the progeny of the cross between Lycopersicon esculentum and L. pennellii, monogenic segregations of 16 isozyme loci were analyzed in an F₂ and two backcross populations of this cross. In the F₂, 9 loci mapping to chromosomes 1, 2, 4, 9, 10 and 12 exhibited skewed segregations and in all cases there was an excess of L. pennellii homozygotes. The genotypic frequencies at all but one locus were at Hardy-Weinberg equilibria. In the backcross populations, all except two loci exhibited normal Mendelian segregations. No post-zygotic selection model could statistically or biologically explain the observed segregation patterns in the F₂ and backcross populations. A pre-zygotic selection model, assuming selective elimination of the male gametophytes during pollen function (i.e., from pollination to karyogamy), could adequately explain the observed segregations in all three populations. The direction of the skewed segregations in the F₂ population was consistent with that expected based on the effects of unilateral incompatibility reactions between the two species. In addition, the chromosomal locations of 5 of the 9 markers that exhibited skewed segregations coincided with the locations of several known compatibility-related genes in tomato. Multigenic unilateral incompatibility reactions between L. esculentum pollen and the stigma or style of L. pennellii (or its hybrid derivatives) are suggested to be the major cause of the skewed segregations in the F₂ progeny of this cross.     

Thermodynamics elucidation of the structural stability of a thermophilic protein

The structural stability of the protein, phycocyanin isolated from two strains of cyanophyta, Synechococcus lividus (thermophile) and Phormidium luridum (mesophile), are investigated by comparative thermal and denaturant unfolding, using differential scanning calorimetry, visible absorption spectrophotometry, and circular dichroism. The thermophilic protein exhibits a much higher temperature and enthalpy of unfolding from the native to the denatured state. The concentration of urea at half-completion of thermal unfolding is essentially the same between the thermophilic and mesophilic proteins; in contrast, the corresponding temperature and the enthalpy of thermal unfolding are much higher for the thermophilic protein. In addition, the concentration of urea at which the non-thermal (denaturant) unfolding of protein is half-completed, as detected by either circular dichroism or absorption spectroscopy, is significantly higher in the thermophilic protein, while the apparent free energy of unfolding only shows a moderate difference between the two proteins. The distinct differences in the enthalpy of thermal unfolding and the free energy of denaturant unfolding are interpreted in terms of a significant entropy change associated with the unfolding of these proteins. This entropy contribution is much higher in the thermophilic protein, and may be derived from its more rigid overall structure that possesses higher internal hydrophobicity and stronger internal packing.     

Exothermic responses of dormant Salix stems during exposure to subzero temperatures

Differential thermal analysis (DTA) was used to determine the exothermic responses in dormant stems and excised lengths of stem of Salix dasyclados Wimmer subjected to artificial freezing treatments.
The presence of ice on the surfaces of intact stems restricted the mechanism of freezing avoidance to temperatures above –4°C. In contrast, excised lengths of stem started to freeze as soon as the ambient temperature fell below –2°C, demonstrating that extracellular ice formation takes place earlier if cut surfaces are present. Exposure of dormant excised lengths of stem to subfreezing temperatures for more than 8 weeks did not alter their nucleation temperature not their exothermic differential responses. Early extracellular crystallisation of freezable cellular water provides conditions that allow dormant Salix dasyclados stems or excised lengths of stem to survive extreme freezing stress.
Crystallisation of extracellular and cellular water took place in the cortex, and did not result in visual damage or reduced survival. This nucleation of extracellular water took place over the same temperature range whether the excised dormant lengths of stem were partly (bark only) or completely thawed. Exposure of dormant tissue to 20°C for up to 24 h did not alter the level of freezing tolerance, nor did it increase the susceptibility of excised lengths of stem to damage by extreme temperature fluctuations.     

FEMALE REPRODUCTIVE EFFORT DEPENDS ON THE DEGREE OF ORNAMENTATION OF THEIR MATES

Sexual selection theory assumes that secondary sexual characters do not influence female reproductive effort. Female animals may invest relatively more in reproduction if they acquire mates of high phenotypic quality, because offspring sired by preferred males may be relatively more viable than offspring sired by less preferred males. Here we report for the first time in a field study that females of the monogamous barn swallow Hirundo rustica adjust their reproductive effort to the attractiveness of their mates. Experimental manipulation of male tail length, which is a trait currently subject to a directional female mating preference, affected the reproductive effort by females in single broods as well as their decision on the seasonal number of clutches. These results, and those of previous experiments, demonstrate that female barn swallows assess the quality of their mates throughout the reproductive season and adjust their reproductive decisions accordingly. This result has important implications for the theory of sexual selection and for the possibility of testing current models of female mate preferences, because the viability of offspring will be confounded by differential reproductive effort.     

Rapid changes in amplification and methylation pattern of genomic DNA in cultured carrot root explants (Daucus carota L.)

Summary Rapid genomic DNA variation due to methylation and copy number alteration was observed in carrot root explants 6 h after inoculation and during a 36-h period of exponential callus growth. De novo methylation and amplification of restricted BspNI fragments of low molecular weight occurred before cell cycle activation and should, therefore, be independent of progression through the S-phase of the cell cycle. Growth regulators seemed to influence the amplification pattern indirectly by regulating cell division activity. In exponentially growing callus tissue the copy number of most of the repetitive fragments was dramatically reduced. It is presumed that this reduction in the copy number of repetitive fragments is characteristic of rejuvenilization. 3-Indole-acetic-acid (IAA) and inositol in the medium increased the degree of unspecific genomic DNA methylation in growing rhizogenic carrot callus tissue in the absence of kinetin, which inhibits root induction at that stage. A possible relation to the induction of rhizogenesis is considered. The observed reduction in number of repetitive restriction fragments and the increase in DNA methylation are gross changes covering the total genome. The results are discussed in relation to the controversy concerning the general biological significance of the methylation and amplification of DNA sequences.     

Identification of New Oligodendrocyte- and Myelin-Specific Genes by a Differential Screening Approach

Abstract: We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte-free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide-synthesizing enzyme UDP-galactose:ceramide galactosyl-transferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T-lymphocyte-specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl-CoA-desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long-chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the β-tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into this complex mechanism.