Study of human chromosome V

Summary The induction of fragile sites on human chromosomes has been demonstrated under various conditions that cause thymidylate stress, including exposure to uridine. In this study, we examined common fragile site expression by initially exposing peripheral lymphocytes to uridine, followed by repair of the fragile sites with media containing various concentrations of thymidine. Lymphocytes were cultured in medium 199 with 2 mM uridine. At 0.5, 1, 2, 3, 8, 10, 12, and 18 h before harvest, the uridine medium was removed and replaced by medium containing thymidine at various concentrations. Our results demonstrate that the effect of uridine on chromosome fragility can be reversed by low concentrations of thymidine (2 M up to 200 M) and the rescuing effect of thymidine can be achieved if the cells were treated prior to 2–3 h before harvest. No repair was found if thymidine was added to culture within 2 h prior to harvesting, suggesting that packing of chromosomes is also an important factor in the expression and repair of fragile sites.     

The effect of sucrose on the levels of abscisic acid, indoleacetic acid and zeatin/zeatin riboside in wheat ears growing in liquid culture

The influence of a varied sucrose supply on grain size and hormonal contents of detached wheat ears ( Triticwn aestivum L. cv. Schirokko) was investigated throughout grain development. In ears led limited amounts, or no sucrose, grain weights in both proximal and distal grain positions of the ear were reduced. Radioimmunoassay for abscisic acid, indoleacetic acid and zeatin/zeatin riboside showed that the changes in the levels of these hormones in grains and bracts were comparable to intact ears when detached ears were well supplied with sucrose. Under conditions of limited sucrose supply, higher abscisic acid levels in the distal and proximal grains of detached ears were found compared to ears supplied with adequate sucrose. Limiting sucrose supply to the ear did not alter the levels of indoleacetic acid or zeatin/zeatin riboside in either the grains or bracts of detached ears.     

Physiological integration in Cassia fasciculata Michx.: inflorescence removal and defoliation experiments

Summary In the annual herb Cassia fasciculata virtually every leaf subtends an axillary inflorescence. We examined the degree to which these leaf-inflorescence units (reproductive nodes) were physiologically independent of each other in the production of flowers, fruits, and seeds. Removal of up to 4 of every 5 inflorescences resulted in substantial increases in fruit and seed production by remaining, intact reproductive nodes. These increases nearly compensated for and manipulated reproductive nodes were associated with different vascular strands. When 2 of every 3 leaves were removed, fruit and seed production were reduced at both intact and defoliated reproductive nodes. Taken together, these results suggest that neighboring reproductive nodes in C. fasciculata are not physiologically independent of one another, and that competition among fruits and seeds for parental resources occurs over several reproductive nodes.Scientific contribution no. 1595 from the New Hampshire Agricultural Experiment Station     

Photosynthetic apparatus of pea thylakoid membranes : response to growth light intensity

We investigated the effect of growth light intensity on the photosynthetic apparatus of pea (Pisum sativum) thylakoid membranes. Plants were grown either in a growth chamber at light intensities that ranged from 8 to 1050 microeinsteins per square meter per second, or outside under natural sunlight. In thylakoid membranes we determined: the amounts of active and inactive photosystem II, photosystem I, cytochrome b/f, and high potential cytochrome b₅₅₉, the rate of uncoupled electron transport, and the ratio of chlorophyll a to b. In leaves we determined: the amounts of the photosynthetic components per leaf area, the fresh weight per leaf area, the rate of electron transport, and the light compensation point. To minimize factors other than growth light intensity that may alter the photosynthetic apparatus, we focused on peas grown above the light compensation point (20-40 microeinsteins per square meter per second), and harvested only the unshaded leaves at the top of the plant. The maximum difference in the concentrations of the photosynthetic components was about 30% in thylakoids isolated from plants grown over a 10-fold range in light intensity, 100 to 1050 microeinsteins per square meter per second. Plants grown under natural sunlight were virtually indistinguishable from plants grown in growth chambers at the higher light intensities. On a leaf area basis, over the same growth light regime, the maximum difference in the concentration of the photosynthetic components was also about 30%. For peas grown at 1050 microeinsteins per square meter per second we found the concentrations of active photosystem II, photosystem I, and cytochrome b/f were about 2.1 millimoles per mol chlorophyll. There were an additional 20 to 33% of photosystem II complexes that were inactive. Over 90% of the heme-containing cytochrome f detected in the thylakoid membranes was active in linear electron transport. Based on these data, we do not find convincing evidence that the stoichiometries of the electron transport components in the thylakoid membrane, the size of the light-harvesting system serving the reaction centers, or the concentration of the photosynthetic components per leaf area, are regulated in response to different growth light intensities. The concept that emerges from this work is of a relatively fixed photosynthetic apparatus in thylakoid membranes of peas grown above the light compensation point.     

Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger

We have isolated AE3, a novel gene expressed primarily in brain neurons and in heart. The predicted AE3 polypeptide shares a high degree of identity with the anion exchange and cytoskeletal binding domains of the erythrocyte band 3 protein. Expression of AE3 cDNA in COS cells leads to chronic cytoplasmic acidification and to chloride- and bicarbonate-dependent changes in intracellular pH, confirming that this gene product is an anion exchanger. Characterization of an AE3 mutant lacking the NH2-terminal 645 amino acids demonstrates that the COOH-terminal half of the polypeptide is both necessary and sufficient for correct insertion into the plasma membrane and for anion exchange activity. The NH2-terminal domain may play a role in regulating the activity of the exchanger and may be involved in the structural organization of the cytoskeleton in neurons.     

Effect of prostaglandin F3 alpha on gastric mucosal injury by ethanol in rats: comparison with prostaglandin F2 alpha

In humans eicosapentaenoic acid can be converted to 3-series prostaglandins (PGF3 alpha, PGI3, and PGE3). Whether 3-series prostaglandins can protect the gastric mucosa from injury as effectively as their 2-series analogs is unknown. Therefore, we compared the protective effects of PGF3 alpha and PGF2 alpha against gross and microscopic gastric mucosal injury in rats. Animals received a subcutaneous injection of either PGF3 alpha or PGF2 alpha in doses ranging from 0 (vehicle) to 16.8 mumol/kg and 30 min later they received intragastric administration of 1 ml of absolute ethanol. Whether mucosal injury was assessed 60 min or 5 min after ethanol, PGF3 alpha was significantly less protective against ethanol-induced damage than PGF2 alpha. These findings indicate that the presence of a third double bond in the prostaglandin F molecule between carbons 17 and 18 markedly reduces the protective effects of this prostaglandin on the gastric mucosa.     

Transient increase in glucose 1,6-bisphosphate in human skeletal muscle during isometric contraction.

Changes in glucose 1,6-bisphosphate and regulators of glucose-1,6-bisphosphate synthase and phosphatase during isometric contraction have been determined. Biopsies were obtained from the quadriceps femoris muscle before and after 20 s of contraction and at fatigue. Glucose 1,6-bisphosphate increased by 35% after 20 s of contraction (P less than 0.001) with no further change at fatigue (P greater than 0.05 versus 20 s). Pi, fructose 1,6-bisphosphate and glycerate 3-phosphate, all inhibitors of the synthase, increased significantly during the first 20 s (P less than 0.05-0.001), whereas muscle pH (decrease in which inhibits synthase) decreased continuously. The decrease in the total adenine nucleotide pool, which is stoichiometric with the increase in IMP (an activator of phosphatase), was not significant after 20 s, but was 15% at fatigue (P less than 0.001). The rapid increase in glucose 1,6-bisphosphate, despite increases in the inhibitors of synthase, suggests that the synthase was activated, possibly by the substrate glycerate 1,3-bisphosphate and/or a yet unknown activator(s). The lack of any further change in glucose 1,6-bisphosphate during the latter part of contraction may be due to concomitant activation of the synthase and phosphatase.     

The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.     

DNA-binding domain of human c-Myc produced in Escherichia coli.

We have identified the domain of the human c-myc protein (c-Myc) produced in Escherichia coli that is responsible for the ability of the protein to bind sequence-nonspecific DNA. Using analysis of binding of DNA by proteins transferred to nitrocellulose, DNA-cellulose chromatography, and a nitrocellulose filter binding assay, we examined the binding properties of c-Myc peptides generated by cyanogen bromide cleavage, of mutant c-Myc, and of proteins that fuse portions of c-Myc to staphylococcal protein A. The results of these analyses indicated that c-Myc amino acids 265 to 318 were responsible for DNA binding and that other regions of the protein (including a highly conserved basic region and a region containing the leucine zipper motif) were not required. Some mutant c-Mycs that did not bind DNA maintained rat embryo cell-cotransforming activity, which indicated that the c-Myc property of in vitro DNA binding was not essential for this activity. These mutants, however, were unable to transform established rat fibroblasts (Rat-1a cells) that were susceptible to transformation by wild-type c-Myc, although this lack of activity may not have been due to their inability to bind DNA.     

Time-resolved fluorescence spectroscopy of lumazine protein from Photobacterium phosphoreum using synchrotron radiation

Time-resolved fluorescence on lumazine protein from Photobacterium phosphoreum was performed with synchrotron radiation as a source of continuously tunable excitation. The experiments yielded structural and dynamic details from which two aspects became apparent. From fluorescence anisotropy decay monitoring of lumazine fluorescence with different excitation wavelengths, the average correlation times were shown to change, which must indicate the presence of anisotropic motion of the protein. A similar study with 7-oxolumazine as the fluorescent ligand led to comparable results. The other remarkable observation dealt with the buildup of acceptor fluorescence, also observed with 7-oxolumazine although much less pronounced, which is caused by the finite energy transfer process between the single donor tryptophan and the energy accepting lumazine derivatives. Global analytical approaches in data analysis were used to yield realistic correlation times and reciprocal transfer rate constants. It was found that the tryptophan residue has a large motional freedom as also reported previously for this protein and for the related protein from P. leiognathi (Lee et al. 1985; Kulinski et al. 1987). The average distance between the tryptophan residue and the ligand donor-acceptor couple has been determined to be 2.7 nm for the same donor and two different acceptors.