An integrated genetic and cytogenetic map for the Mediterranean fruit fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>, based on microsatellite and morphological markers

A genetic map based on microsatellite polymorphisms and visible mutations of the Mediterranean fruit fly (medfly), Ceratitis capitata is presented. Genotyping was performed on single flies from several backcross families. The map is composed of 67 microsatellites and 16 visible markers distributed over four linkage groups. Fluorescence in situ hybridization of selected microsatellite markers on salivary gland polytene chromosomes allowed the alignment of these groups to the second, fourth, fifth and sixth chromosome. None of the markers tested showed segregation either with the X or the third chromosome. However, this map constitutes a substantial starting point for a detailed genetic map of C. capitata. The construction of an integrated map covering the whole genome should greatly facilitate genetic studies and future genome sequence projects of the species.     

Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14 d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.     

An ancient look at UCP1

Brown adipose tissue serves as a thermogenic organ in placental mammals to defend body temperature in the cold by nonshivering thermogenesis. The thermogenic function of brown adipose tissue is enabled by several specialised features on the organ as well as on the cellular level, including dense sympathetic innervation and vascularisation, high lipolytic capacity and mitochondrial density and the unique expression of uncoupling protein 1 (UCP1). This mitochondrial carrier protein is inserted into the inner mitochondrial membrane and stimulates maximum mitochondrial respiration by dissipating proton-motive force as heat. Studies in knockout mice have clearly demonstrated that UCP1 is essential for nonshivering thermogenesis in brown adipose tissue. For a long time it had been presumed that brown adipose tissue and UCP1 emerged in placental mammals providing them with a unique advantage to survive in the cold. Our subsequent discoveries of UCP1 orthologues in ectotherm vertebrates and marsupials clearly refute this presumption. We can now initiate comparative studies on the structure–function relationships in UCP1 orthologues from different vertebrates to elucidate when during vertebrate evolution UCP1 gained the biochemical properties required for nonshivering thermogenesis.     

Functional analysis of histone methyltransferase g9a in B and T lymphocytes

Lymphocyte development is controlled by dynamic repression and activation of gene expression. These developmental programs include the ordered, tissue-specific assembly of Ag receptor genes by V(D)J recombination. Changes in gene expression and the targeting of V(D)J recombination are largely controlled by patterns of epigenetic modifications imprinted on histones and DNA, which alter chromatin accessibility to nuclear factors. An important component of this epigenetic code is methylation of histone H3 at lysine 9 (H3K9me), which is catalyzed by histone methyltransferases and generally leads to gene repression. However, the function and genetic targets of H3K9 methyltransferases during lymphocyte development remain unknown. To elucidate the in vivo function of H3K9me, we generated mice lacking G9a, a major H3K9 histone methyltransferase, in lymphocytes. Surprisingly, lymphocyte development is unperturbed in G9a-deficient mice despite a significant loss of H3K9me2 in precursor B cells. G9a deficiency is manifest as modest defects in the proliferative capacity of mature B cells and their differentiation into plasma cells following stimulation with LPS and IL-4. Precursor lymphocytes from the mutant mice retain tissue- and stage-specific control over V(D)J recombination. However, G9a deficiency results in reduced usage of Iglambda L chains and a corresponding inhibition of Iglambda gene assembly in bone marrow precursors. These findings indicate that the H3K9me2 epigenetic mark affects a highly restricted set of processes during lymphocyte development and activation.     

Structural basis for natural lactonase and promiscuous phosphotriesterase activities

Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 Å resolution) and in complex with a quorum-sensing lactone mimic at 2.0 Å resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.     

The opposite effects of short- and long-term salt loading on pituitary adrenal axis activity in rats.

Osmotic stimulation has been shown to modify corticotropin responsiveness. We compared the effects of short- and long-term salt loading on pituitary-adrenal activity in control rats receiving tap water and rats submitted to salt loading for 1 day (S1) or 8 days (S8). Corticosterone (B) and adrenocorticotropic hormone (ACTH) plasma levels were determined at 8 a. m. under basal conditions or after immobilization stress for 15 min or corticotropin-releasing hormone (CRH) stimulation. S1 rats showed a similar ACTH response to immobilization, but an increased CRH response. In contrast, S8 rats showed blunted responses after immobilization or CRH stimulation. To evaluate the circadian variation of this inhibitory effect on the stress in the S8 group, immobilization was also performed at 8 p. m. Plasma ACTH and B levels under resting conditions were higher at 8 p. m. than 8 a. m. (p < 0.05) in control and S8 rats. The ACTH response to immobilization in the S8 group was lower than control at both 8 a. m. and 8 p. m. (p < 0.05); however, this reduction was more evident the morning, resulting in an inversion of the diurnal pattern with a higher ACTH response at 8 p. m. In conclusion, short osmotic stimulation results in an increased pituitary response to CRH, whereas prolonged stimulation decreases the pituitary response to CRH and immobilization, showing an interaction between osmoregulation and hypothalamus-pituitary-adrenocortical activity.     

Scavenger receptor class B type I is expressed in cultured keratinocytes and epidermis. Regulation in response to changes in cholesterol homeostasis and barrier requirements.

Cholesterol is a key lipid in the stratum corneum, where it is critical for permeability barrier homeostasis. The epidermis is an active site of cholesterol synthesis, but inhibition of epidermal cholesterol synthesis with topically applied statins only modestly affects epidermal permeability barrier function, suggesting a possible compensatory role for extraepidermal cholesterol. Scavenger receptor class B type I (SR-BI) is a recently described cell surface receptor for high density lipoproteins (HDL) that mediates the selective uptake of cholesterol esters from circulating HDL. In the present study, we demonstrate that SR-BI is present in cultured human keratinocytes and that calcium-induced differentiation markedly decreases SR-BI levels. Additionally, the cell association of [(3)H]cholesterol-labeled HDL decreased in differentiated versus undifferentiated keratinocytes. Furthermore, the inhibition of cholesterol synthesis with simvastatin resulted in a 3-4-fold increase in both SR-BI mRNA and protein levels, whereas conversely, addition of 25-hydroxycholesterol suppressed SR-BI levels by approximately 50%. SR-BI mRNA is also expressed in murine epidermis, increasing by 50% in parallel with cholesterol requirements following acute barrier disruption. Because the increase is completely blocked by occlusion with a vapor-impermeable membrane, changes in epidermal SR-BI expression are regulated specifically by barrier requirements. Lastly, using immunofluorescence we demonstrated that SR-BI is present in human epidermis predominantly in the basal layer and increases following barrier disruption. In summary, the present study demonstrates first that SR-BI is expressed in keratinocytes and regulated by cellular cholesterol requirements, suggesting that it plays a role in keratinocyte cholesterol homeostasis. Second, the increase in SR-BI following barrier disruption suggests that SR-BI expression increases to facilitate cholesterol uptake leading to barrier restoration.     

A new series of 3-phenylcoumarins as potent and selective MAO-B inhibitors

6-Methyl-3-phenylcoumarins 3–6 were designed, synthesized and evaluated as monoamine oxidase A and B (MAO-A and MAO-B) inhibitors. The synthesis of these new compounds (resveratrol–coumarin hybrids) was carried out with good yield by a Perkin reaction, from the 5-methylsalicylaldehyde and the corresponding phenylacetic acid. They show high selectivity to the MAO-B isoenzyme, with IC₅₀ values in the nanomolar range. Compound 5 is the most active compound and is several times more potent and selective than the reference compound, R-(?)-deprenyl.     

No Need to Discriminate? Reproductive Diploid Males in a Parasitoid with Complementary Sex Determination

Diploid males in hymenopterans are generally either inviable or sterile, thus imposing a severe genetic load on populations. In species with the widespread single locus complementary sex determination (sl-CSD), sex depends on the genotype at one single locus with multiple alleles. Haploid (hemizygous) individuals are always males. Diploid individuals develop into females when heterozygous and into males when homozygous at the sex determining locus. Our comparison of the mating and reproductive success of haploid and diploid males revealed that diploid males of the braconid parasitoid Cotesia glomerata sire viable and fertile diploid daughters. Females mated to diploid males, however, produced fewer daughters than females mated to haploid males. Nevertheless, females did not discriminate against diploid males as mating partners. Diploid males initiated courtship display sooner than haploid males and were larger in body size. Although in most species so far examined diploid males were recognized as genetic dead ends, we present a second example of a species with sl-CSD and commonly occurring functionally reproductive diploid males. Our study suggests that functionally reproductive diploid males might not be as rare as hitherto assumed. We argue that the frequent occurrence of inbreeding in combination with imperfect behavioural adaptations towards its avoidance promote the evolution of diploid male fertility.