Stress response in Drosophila melanogaster strain inactive with decreased tyramine and octopamine contents

Juvenile hormone hydrolysis, tyrosine decarboxylase activity, dopamine content and fitness (viability and fertility) were studied under normal and stress conditions in the adults of Drosophila melanogaster inactive strain carrying a mutation that decreases tyrosine decarboxylase activity and results in the lower contents of tyramine and octopamine. A sexual dimorphism of tyrosine decarboxylase activity, dopamine level and survival under heat stress in inactive flies was found. inactive adults showed higher dopamine levels and lower survival levels under heat stress as compared to wild type (Canton S) adults. Juvenile hormone degradation is decreased in young and increased in mature inactive females as compared to wild type. Fertility of the inactive strain did not differ from that of wild type strain under normal conditions, but after heat exposure the dynamics of its restoration was different. inactive females were found to develop the stress reaction, with juvenile hormone degradation, tyrosine decarboxylase activity, dopamine content and fertility levels used as the reaction indicators.     

Possible role of octopamine and tyramine in the antihypertensive and antidepressant effects of tyrosine

The administration of a dose of 200 mg/kg of tyrosine (as either the free amino acid or the ethyl ester) increased the 24-hour excretion of p-hydroxyphenethyleneglycol (p-HPG) and p-hydroxyphenylethanol, metabolites of octopamine and tyramine, by 147 and 50%, respectively. One hour after this dose of tyrosine, brain levels of p-HPG and p-hydroxyphenylacetic acid (p-HPA), another metabolite of tyramine, were increased by 82 and 196%, respectively. Pretreatment with Ro4-4602, a peripheral decarboxylase inhibitor, reduced by 50% the tyrosine-induced increases in brain p-HPA levels, suggesting that tyramine was partially formed in the brain parenchyma. Tyrosine caused only slight, but non-significant increases in brain levels of catecholamine metabolites. These results suggest that tyrosine-induced increases in the production of tyramine and octopamine in brain may account for some of the effects of tyrosine, such as its antihypertensive and reported antidepressant properties.     

Two Drosophila melanogaster tropomyosin genes: structural and functional aspects.

We compared the structure and function of the two Drosophila melanogaster tropomyosin genes. The most striking structural aspect was their size disparity. Codons 1 through 257 of gene 2 occupied 833 nucleotides and contained only one intron, whereas the corresponding region of gene 1 occupied 17.5 kilobases and was interrupted by eight introns. The intron-exon arrangement of gene 1 reflected evolutionary expansion of tropomyosin via 42- and 49-residue duplications, which are probably actin-binding domains. Functionally, gene 1 was considerably more complex than gene 2; it was active in both muscle and nonmuscle cell lineages, had at least five variable exons, and specified a minimum of five developmentally regulated isoforms. Two of these isoforms, which accumulated only in flight muscles, were unprecedented fusion proteins in which the tropomyosin sequence was joined to a carboxy-terminal proline-rich domain.     

Two distinct roles for two functional cobaltochelatases (CbiK) in Desulfovibrio vulgaris hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses a large number of porphyrin-containing proteins whose biosynthesis is poorly characterized. In this work, we have studied two putative CbiK cobaltochelatases present in the genome of D. vulgaris. The assays revealed that both enzymes insert cobalt and iron into sirohydrochlorin, with specific activities with iron lower than that measured with cobalt. Nevertheless, the two D. vulgaris chelatases complement an E. coli cysG mutant strain showing that, in vivo, they are able to load iron into sirohydrochlorin. The results showed that the functional cobaltochelatases have distinct roles with one, CbiK(C), likely to be the enzyme associated with cytoplasmic cobalamin biosynthesis, while the other, CbiK(P), is periplasmic located and possibly associated with an iron transport system. Finally, the ability of D. vulgaris to produce vitamin B 12 was also demonstrated in this work.     

Prepatterning the Drosophila notum: the three genes of the iroquois complex play intrinsically distinct roles

The Drosophila thorax exhibits 11 pairs of large sensory organs (macrochaetes) identified by their unique position. Remarkably precise, this pattern provides an excellent model system to study the genetic basis of pattern formation. In imaginal wing discs, the achaete-scute proneural genes are expressed in clusters of cells that prefigure the positions of each macrochaete. The activities of prepatterning genes provide positional cues controlling this expression pattern. The three homeobox genes clustered in the iroquois complex (araucan, caupolican and mirror) are such prepattern genes. mirror is generally characterized as performing functions predominantly different from the other iroquois genes. Conversely, araucan and caupolican are described in previous studies as performing redundant functions in most if not all processes in which they are involved. We have addressed the question of the specific role of each iroquois gene in the prepattern of the notum and we clearly demonstrate that they are intrinsically different in their contribution to this process: caupolican and mirror, but not araucan, are required for the neural patterning of the lateral notum. However, when caupolican and/or mirror expression is reduced, araucan loss of function has an effect on thoracic bristles development. Moreover, the overexpression of araucan is able to rescue caupolican loss of function. We conclude that, although retaining some common functionalities, the Drosophila iroquois genes are in the process of diversification. In addition, caupolican and mirror are required for stripe expression and, therefore, to specify the muscular attachment sites prepattern. Thus, caupolican and mirror may act as common prepattern genes for all structures in the lateral notum.     

Drosophila female courtship and mating behaviors: sensory signals, genes, neural structures and evolution

Interest in Drosophila courtship behavior has a long-standing tradition, starting with the works by Sturtevant in 1915, and by Bastock and Manning in the 50s. The neural and genetic base of Drosophila melanogaster courtship behavior has made big strides in recent years, but the studies on males far outnumber those on females. Recent technical developments have made it possible to begin to unravel the biological substrates underlying the complexity of Drosophila female sexual behavior and its decisive effect on mating success. The present review focus more on the female side and summarizes the sensory signals that the male sends, using multiple channels, and which neural circuits and genes are mediating sex-specific behavioral responses.     

The genetics of dopa decarboxylase in Drosophila melanogaster. III. Effects of a temperature sensitive dopa decarboxylase deficient mutation on female fertility

The mutation Ddc^ts1 effects female sterility when homozygous, hemizygous, or heterozygous over a series of Ddc null alleles (Ddc^x) indicating that some aspect of Ddc gene function is necessary for female fertility. Ovary transplant experiments demonstrate that the female sterility phenotype is ovary autonomous. Two to 3% of the total DDC activity measurable in newly hatched females is localized in their previtellogenic ovaries. The degree to which females heterozygous for Ddc^ts1 over different Ddc null alleles are fertile at 22°C reflects a continuous spectrum of allelic complementation similar to that observed for the effects of these genotypes on viability at 30°C. Fertility of all the Ddc^ts1/Ddc^x females tested is significantly depressed at 30 vis-a-vis 22°C providing evidence that it is the DDC enzyme activity itself which is required for female fertility. Ddc^ts1/Ddc^ts1 homozygous and Ddc^ts1/Df hemizygous females are nonconditionally, completely sterile at 18, 20, 22, 25, and 30°C. Although all homo- and hemizygous females do lay some eggs, no evidence of embryogenesis or fertilization has ever been detected. The absolute, nonconditional sterility of Ddc^ts1 homo- and hemizygous females stands in stark contrast to the conventional temperature dependent effects of these same genotypes on viability and to the temperature sensitive effects of Ddc^ts1/Ddc^x heterozygous females on both fertility and viability. Reasons for these tissue-specific and genotypic differences are discussed.     

Three distinct roles for notch in Drosophila R7 photoreceptor specification

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.     

Fixed and dilutable benefits: female choice for good genes or fertility

Benefits accruing to females who exercise mate choice have been defined to be either 'direct' or 'indirect'. We suggest an alternative distinction: benefits can be considered 'fixed', meaning they are on average equal to all females mating with the same male (e.g. good genes' benefits) or 'dilutable', meaning they are shared between females mating with the same male, so that the more mates a male has, the lower the average benefit to each (e.g. fertility benefits or many forms of direct benefit). Using a simple model, we show that this distinction has a major effect on the form of female preference. We predict that mating skew will be far greater in species where the benefits are fixed when compared with those where the benefits are dilutable.     

Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC xTYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.     

Expression of tryptophan decarboxylase and tyrosine decarboxylase genes in tobacco results in altered biochemical and physiological phenotypes

The substrate specificity of tryptophan (Trp) decarboxylase (TDC) for Trp and tyrosine (Tyr) decarboxylase (TYDC) for Tyr was used to modify the in vivo pools of these amino acids in transgenic tobacco. Expression of TDC and TYDC was shown to deplete the levels of Trp and Tyr, respectively, during seedling development. The creation of artificial metabolic sinks for Trp and Tyr also drastically affected the levels of phenylalanine, as well as those of the non-aromatic amino acids methionine, valine, and leucine. Transgenic seedlings also displayed a root-curling phenotype that directly correlated with the depletion of the Trp pool. Non-transformed control seedlings could be induced to display this phenotype after treatment with inhibitors of auxin translocation such as 2,3,5-triiodobenzoic acid or N-1-naphthylphthalamic acid. The depletion of aromatic amino acids was also correlated with increases in the activities of the shikimate and phenylpropanoid pathways in older, light-treated transgenic seedlings expressing TDC, TYDC, or both. These results provide in vivo confirmation that aromatic amino acids exert regulatory feedback control over carbon flux through the shikimate pathway, as well as affecting pathways outside of aromatic amino acid biosynthesis.     

Distinct roles for two receptor tyrosine kinases in epithelial branching morphogenesis in Drosophila

Branching morphogenesis is a widespread mechanism used to increase the surface area of epithelial organs. Many signaling systems steer development of branched organs, but it is still unclear which cellular processes are regulated by the different pathways. We have used the development of the air sacs of the dorsal thorax of Drosophila to study cellular events and their regulation via cell-cell signaling. We find that two receptor tyrosine kinases play important but distinct roles in air sac outgrowth. Fgf signaling directs cell migration at the tip of the structure, while Egf signaling is instrumental for cell division and cell survival in the growing epithelial structure. Interestingly, we find that Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct migration, suggesting that both cytoskeletal and nuclear events are downstream of receptor activation. Ras and the Mapk pathway are also needed for Egf-regulated cell division/survival, but Pointed is dispensable.     

Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading

Focal adhesion (FA) formation is induced by extracellular matrix-stimulated integrin clustering and activation of receptors for diffusible factors. Leupaxin (LPXN) is a member of the paxillin family of FA proteins expressed in many cancer cell lines. We found activation of gastrin-releasing peptide receptor (GRPr) by bombesin (BN) stimulated LPXN translocation from cytoplasm to FAs. Using mutagenesis, we identified LIM3 as the primary FA targeting domain for LPXN and showed BN-induced LPXN tyrosine phosphorylation on residues 22, 62 and 72. A LIM3 point mutant of LPXN failed to target to FAs and had no BN-stimulated tyrosine phosphorylation. Conversely, a non-phosphorylatable mutant (Y22/62/72F) translocated to FAs after BN addition. Stimulation of FA formation using vinblastine also induced LPXN translocation and tyrosine phosphorylation. Therefore, dynamic LPXN tyrosine phosphorylation requires translocation to FAs. LPXN and paxillin had opposite roles in adhesion to collagen I (CNI) in MDA-MB-231 breast cancer cells. LPXN siRNA stimulated whereas paxillin siRNA inhibited cell adhesion. Knockdown of both LPXN and paxillin behaved similarly to paxillin knockdown alone, suggesting LPXN’s function in adhesion might depend on paxillin. Additionally, LPXN regulated cell spreading on CNI but not on fibronectin whereas paxillin knockdown suppressed spreading on both substrates. These results demonstrate that although LPXN and paxillin’s FA targeting and tyrosine phosphorylation are similar, each protein has distinct functions.     

Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading

Focal adhesion (FA) formation is induced by extracellular matrix-stimulated integrin clustering and activation of receptors for diffusible factors. Leupaxin (LPXN) is a member of the paxillin family of FA proteins expressed in many cancer cell lines. We found activation of gastrin-releasing peptide receptor (GRPr) by bombesin (BN) stimulated LPXN translocation from cytoplasm to FAs. Using mutagenesis, we identified LIM3 as the primary FA targeting domain for LPXN and showed BN-induced LPXN tyrosine phosphorylation on residues 22, 62 and 72. A LIM3 point mutant of LPXN failed to target to FAs and had no BN-stimulated tyrosine phosphorylation. Conversely, a non-phosphorylatable mutant (Y22/62/72F) translocated to FAs after BN addition. Stimulation of FA formation using vinblastine also induced LPXN translocation and tyrosine phosphorylation. Therefore, dynamic LPXN tyrosine phosphorylation requires translocation to FAs. LPXN and paxillin had opposite roles in adhesion to collagen I (CNI) in MDA-MB-231 breast cancer cells. LPXN siRNA stimulated whereas paxillin siRNA inhibited cell adhesion. Knockdown of both LPXN and paxillin behaved similarly to paxillin knockdown alone, suggesting LPXN''s function in adhesion might depend on paxillin. Additionally, LPXN regulated cell spreading on CNI but not on fibronectin whereas paxillin knockdown suppressed spreading on both substrates. These results demonstrate that although LPXN and paxillin''s FA targeting and tyrosine phosphorylation are similar, each protein has distinct functions.Key words: focal adhesion, tyrosine phosphorylation, bombesin, adhesion, spreading