Pituitary adenylate cyclase-activating peptide: a pivotal modulator of steroid-induced reproductive behavior in female rodents

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates the secretion of GnRH into the hypothalamic hypophysial portal system and sensitizes the pituitary for release of hormones that trigger ovulation. Because reproductive behavior is synchronized with GnRH release, the present study was undertaken to determine whether PACAP in the ventromedial nucleus (VMN) plays a role in receptivity. To this end, we used rat and mouse reproductive behavioral models to determine the biological relationship between PACAP and steroid receptor function in females. We provide evidence for the requirement of PACAP in the VMN for progesterone (P)-dependent sexual behavior in estrogen (E)-primed females. We clarify the biological and molecular mechanisms of PACAP activity by showing 1) that inhibition of endogenous PACAP suppresses P receptor (PR)-dependent sexual behavior facilitated by the steroid P or D1-like agonist SKF38393 and 2) that PR, steroid receptor coactivators-1 and -2, and new protein synthesis are essential for ligand independent PACAP-facilitated behavior. These findings are consistent with convergence of PACAP-mediated cellular signals on PR for genomic activation and subsequent behavioral changes. Further, we show that steroids regulate both endogenous PACAP mRNA in the VMN and immunoreactive PACAP in the medial basal hypothalamus and cerebral spinal fluid for ligand-dependent, steroid receptor-dependent receptivity. The present findings delineate a novel, steroid-dependent mechanism within the female hypothalamus by which the neuropeptide PACAP acts as a feed-forward, paracrine, and/or autocrine factor for synchronization of behavior coordinate with hypothalamic control of ovulation.     

Cuticular wax profiles of leaves of some traditionally used African Bignoniaceae

Bignoniaceae, Newbouldia laevis, Markhamia acuminata, Spathodea campanulata and Kigelia africana were analysed by GC-MS. The principal constituents were represented by a homologous series of n-alkanes (C23-C33), n-alcohols (C18-C30) and related carboxylic acids (C16-C36). For N. laevis and M. acuminata, ursolic and oleanolic acid were the most abundant wax components (52 and 60%, respectively), followed by the C29, the C31 and the C33 n-alkanes. The predominant components of S. campanulata were n-alcohols (35%), with octacosanol and triacontanol as the most abundant ones, while K. africana is distinguished from these three members by the conspicuous absence of triterpenoic acids and the predominance of n-alkanes (70%) with hentriacontane and tritriacontane as the main representatives. Other notable constituents were sterols, albeit present in trace amounts. The wax profiles are discussed in terms of taxonomic characters.     

Expression of collagen type I and type II in consecutive stages of human osteoarthritis

In normal hyaline cartilage the predominant collagen type is collagen type II along with its associated collagens, for example, types IX and XI, produced by normal chondrocytes. In contrast, investigations have demonstrated that in vitro a switch from collagen type II to collagen type I occurs. Some authors have detected collagen type I in osteoarthritic cartilage also in vivo, especially in late stages of osteoarthritis, while others have not. In the light of these diverging results, we have attempted to elucidate which type of collagen, type I and/or type II, is synthesized in the consecutive stages of human osteoarthritis. We performed in situ hybridization and immunohistochemistry with cartilage tissue samples from patients suffering from various stages of osteoarthritis. Furthermore, we quantitated our results on the gene expression of collagen type I and type II with the help of real-time PCR. We found that with the progression of the disease not only collagen type II, but also increasing amounts of collagen type I mRNA were produced. This supports the conclusion that collagen type I gradually becomes one of the factors involved in the pathogenesis of osteoarthritis.     

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.     

ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels

During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (DeltaG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (DeltaG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl- from guard cells and a rapid but confined reduction in stomatal aperture.     

Subfascial periareolar augmentation mammaplasty

Subfascial placement of implants was introduced 3 years ago. Collected data reveal very promising short-term and long-term results in comparison with subglandular and subpectoral positioned implants. The clinical experiences of 69 breast augmentations in the subfascial position are reported. The indications for this technique are proposed. The incidence of complications is described from clinical experiences and compared with that for other methods. From January of 1998 through May of 2002, 328 patients underwent periareolar augmentation mammaplasty; 105 patients had a subglandular mammaplasty, 154 patients had a subpectoral mammaplasty, and from August of 1999 through May of 2002, 69 patients had a subfascial augmentation mammaplasty. The mean postoperative follow-up time was 3.6 years in the subglandular group, 3.5 years in the subpectoral group, and 2.9 years in the subfascial group. In comparing the results of the subglandular augmentation group with those of the subpectoral and subfascial augmentation groups, the total rate of complications diminished significantly. The long-term complications of severe capsular contracture, rippling, and nipple sensation and numbness in subglandular augmentation mammaplasty could be significantly reduced (p < 0.05). The subfascial augmentation mammaplasty unites all the advantages of the subpectoral augmentation mammaplasty but eliminates the disadvantages of increased postoperative discomfort and disturbing muscle movement of the breast.     

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

Separation of fluorescence signals from Ca2+ and NADH during cardioplegic arrest and cardiac ischemia

Determinations of intracellular [Ca(2+)](i) during ischemia using fluorescent indicators are hampered by overlapping cellular autofluorescence (AF), which largely depends on NADH. If Ca(2+) is to be determined under different kinds of ischemia, signal separation merits special attention. We used triple wavelength excitation fluorescence to separate autofluorescence from [Ca(2+)]-dependent fura-2 fluorescence. Excitation at 360 nm served as third, Ca(2+)-insensitive wavelength. Using an appropriate evaluation procedure, we separated Ca(2+)-dependent signals from autofluorescence which is semiquantitatively associated with NADH, an indicator of the cellular redox state. We compared changes of [Ca(2+)](i) in isolated hearts during ischemia following cardioplegic arrest with those after transient stop of nutritive perfusion. We observed [Ca(2+)] transients in spontaneously beating hearts, persisting during ischemic episodes, and an increase of mean [Ca(2+)](i). In contrast, cardioplegic arrest stopped periodical [Ca(2+)](i) transients and heart beats simultaneously. [Ca(2+)](i) remained at diastolic values, tended to decrease during the first minutes of cardioplegic arrest and then increased slowly. Autofluorescence increased under both conditions. During ischemia, this increase was faster than in cardioplegia experiments. It started after the last heart beat despite persisting perfusion. Our measurements demonstrate that rhythmical heart beat is essential for sufficient perfusion. Reduced [Ca(2+)](i) under cardioplegic arrest may influence metabolism.     

Combining plant- and soil-dwelling predatory mites to optimise biological control of thrips

The efficiency of a natural enemy combination compared to a single species release for the control of western flower thrips (WFT) Frankliniella occidentalis (Pergande) on cucumber plants was investigated. Since a large part of F occidentalis seems to enter the soil passage, a joint release of the plant-inhabiting predatory mite Amblyseius cucumeris (Oudemans) that feeds on thrips first-instar larvae and the soil-dwelling predatory mite Hypoaspis aculeifer (Canestrini) that preys on thrips pupae in the ground might offer a promising approach for a holistic control strategy. Therefore, two sets of experiments were conducted in cooperation with a commercial vegetable grower where the plants in plots were infested with a defined number of larval and adult F occidentalis. Two species of natural enemies were released either synchronously or solely, and their efficacy was compared to control plots devoid of antagonists. In both experiments, the predatory mites were released twice with a density of 46 A. cucumeris/m2, and 207 H. aculeifer/m2 (low-density) in the first experiment and 528 H. aculeifer/m2 (high-density) in the second one. Population growth of all arthropod species on the plants and in the soil was quantified at regular intervals and included all soil-dwelling mites and alternative preys present in the substrate. The results showed that H. aculeifer alone had a significant impact on thrips population development only when released at high-densities, but competence was lower compared to the other antagonist treatments. The impact of A. cucumeris alone and A. cucumeris & H. aculeifer combined was similar. Thus, the pooled exploitation of natural enemies did not boost thrips control compared to the single species application of A. cucumeris (non-additive effect), which could be explained by resource competition between both predatory mite species. Species number and population size in the soil of the experimental plots both showed a high variability, a possible consequence of their interaction with released soil-dwelling predatory H. aculeifer mites. The impact of resource competition and presence of alternative preys on thrips biological control is exhaustively discussed. From our study, we can extract the subsequent conclusions: (1) the combined use of H. aculeifer and A. cucumeris cannot increase thrips control on cucumber compared to the release of A. cucumeris alone, but the overall reliability of thrips biological control might be enhanced, (2) the availability of alternative preys seemed to affect the thrips predation rate of H. aculeifer, and (3) the impact of naturally occurring soil predatory mites on the control of WFT seemed to be partial.     

Gamma-COP appendage domain - structure and function

COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the β-, γ-, δ- and ζ-COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the α-, β'- and ε-COP subunits. Here, we present the structure of the appendage domain of γ-COP and show that it has a similar overall fold as the α-appendage of AP2. Again, like the α-appendage the γ-COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian γ-COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of γ-COP, Sec21p, a second binding site is proposed to exist on the γ-COP appendage that interacts with the α,β',ε COPI subcomplex.