Comparison of prostaglandin F2alpha,bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.     

Thin-layer cytology findings of small cell carcinoma of the lower female genital tract. Review of three cases with molecular analysis

OBJECTIVE: To describe the thin-layer cytology findings of small cell carcinoma of the low female genital tract, with histologic correlation and human papillomavirus (HPV) genotyping. STUDY DESIGN: The authors reviewed the clinical findings, thin-layer cytology and histologic features of small cell carcinoma of the lower female genital tract (cervix or vagina) occurring in three postmenopausal Chinese women at Pamela Youde Nethersole Eastern Hospital, Hong Kong, over a four-year period, from January 1998 to December 2001. Molecular techniques for HPV screening and genotyping using the polymerase chain reaction and restriction fragment length polymorphism were employed on the cytologic specimens. RESULTS: The thin-layer preparations were of moderate to high cellularity. There were loose aggregates of or isolated small round cells with a high nuclear/cytoplasmic ratio, thin but irregular nuclear membrane, hyperchromatic nuclei, inconspicuous nucleoli and scanty cytoplasm. Tumor cell cannibalism was commonly found. Small groups of tumor cells with nuclear molding were noted. There was also obvious tumor diathesis in the background. The necrotic debris was admixed with isolated small round cells, apoptotic bodies and nuclear dust. Associated koilocytosis or squamous intraepithelial lesions were not seen. Histologic examination of the tumor biopsies showed classic features of small cell carcinoma associated with squashing artifacts and vascularized stroma. Molecular analysis revealed the presence of HPV DNA (either type 18 or 16) in all the three liquid-based cytology samples. CONCLUSION: While the cytomorphologic features of small cell carcinoma of the cervix or vagina in thin-layer preparations are slightly different from those in conventional smears, due mainly to the absence of smearing effect, recognition of the subtle but characteristic appearance can enhance the accuracy of the cytologic diagnosis. The association between HPV and primary small cell carcinoma of the lower female genital tract was confirmed by this study.     

The establishment of Caenorhabditis elegans germline pattern is controlled by overlapping proximal and distal somatic gonad signals

We investigated the control of proliferation and differentiation in the larval Caenorhabditis elegans hermaphrodite germ line through analysis of glp-1 and lag-2 mutants, cell ablations, and ultrastructural data. After the first several rounds of germ cell division, GLP-1, a receptor of the LIN-12/Notch family, governs germline proliferation. We analyzed the proximal proliferation (Pro) phenotype in glp-1(ar202) and found that initial meiosis was delayed and spatially mispositioned. This is due, at least in part, to a heightened response of the mutant GLP-1 receptor to multiple sources of the somatic ligand LAG-2, including the proximal somatic gonad. We investigated whether proximal LAG-2 affects germline proliferation in the wild type. Our results indicate that (1) LAG-2 is necessary for GLP-1-mediated germline proliferation and prevention of early meiosis, and (2) several distinct anatomical sources of LAG-2 in the larval somatic gonad functionally overlap to promote proliferation and prevent early meiosis. Ultrastructural studies suggest that mitosis is not restricted to areas of direct DTC-germ line contact and that the germ line shares a common cytoplasm in larval stages. We propose that downregulation of the GLP-1 signaling pathway in the proximal germ line at the time of meiotic onset is under tight temporal and spatial control.     

gp38k (CHI3L1) is a novel adhesion and migration factor for vascular cells

gp38k (CHI3L1) is a secreted heparin-binding glycoprotein whose expression, in vitro, is associated with vascular smooth muscle cell (VSMC) migration and invasion into the underlying gelatinous matrix. gp38k is expressed at high levels in postconfluent "nodular" VSMC cultures and at low levels in subconfluent proliferating cultures. In vivo, expression of gp38k homologs is high in regions of tissue remodeling and now has been detected in atherosclerotic plaques and in the developing heart. We tested the hypothesis that gp38k functions to modulate VSMC adhesion and migration. By use of modified Boyden chambers, gp38k at a concentration as low as 1 ng/ml has profound effects on VSMC migration but little or no effect on fibroblast migration. In addition, gp38k adsorbed to polystyrene surfaces directly promotes VSMC attachment and spreading. Attachment is inhibited in the presence of affinity-purified anti-gp38k or 10 mM EDTA. These results establish that gp38k is a new vascular cell adhesion and migration factor that may have a role in processes leading to vascular occlusion and heart development. gp38k may interact with VSMC via an EDTA-sensitive mechanism consistent with integrin mediated cell-matrix interaction.     

Endocrine correlates of intra-specific variation in the mating system of the St. Peter's fish (Sarotherodon galilaeus)

The Challenge Hypothesis postulates that androgen levels are a function of the social environment in which the individual is living. Thus, it is predicted that in polygynous males that engage in social interactions, androgen levels should be higher than in monogamous animals that engage in parental care. In this study, we tested this hypothesis at the intra-specific level using a teleost species, Sarotherodon galilaeus, which exhibits a wide variation in its mating system. Experimental groups of individually marked fish were formed in large ponds with different operational sex-ratios (OSR) to study the effects of partner availability on blood plasma levels of sex steroids [11-ketotestosterone (11-KT), testosterone (T), and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P)] and gonadosomatic index (GSI). Polygyny mostly occurred in the female biased OSR groups. 17,20beta-P and gonadosomatic index did not differ among OSR groups. However, 11-KT was high in male biased OSR and positively correlated with aggressive challenges, thereby supporting the central postulate of the Challenge Hypothesis. The results of T were the inverse of those of 11-KT, probably because 11-KT is metabolized from T. 11-KT levels of polygynous males did not differ neither from those of monogamous males, nor from those of males that participated in parental care. These results do not support the expected relationships between polygyny, parental care, and androgen levels. The differences from expectations for 11-KT may be related to the fact that in S. galilaeus, the mating and the parenting phase are not clearly separated and thus, males may still fight and court while they are brooding.     

Yersinia enterocolitica invasin triggers phagocytosis via beta1 integrins, CDC42Hs and WASp in macrophages

The Yersinia outer surface protein invasin binds to β1 integrins on target cells and has been shown to trigger phagocytic uptake by macrophages. Here, we investigated the role of the actin regulator Wiskott–Aldrich syndrome protein (WASp), its effector the Arp2/3 complex and the Rho-GTPases CDC42Hs, Rac and Rho in invasin/β1 integrin-triggered phagocytosis. During uptake of invasin-coated latex beads, the α5β1 integrin, WASp and the Arp2/3 complex were recruited to the developing actin-rich phagocytic cups in primary human macrophages. Blockage of β1 integrins by specific antibodies, inhibition of Arp2/3 function by microinjection of inhibitors or the use of WASp knockout macrophages inhibited phagocytic cup formation and uptake. Furthermore, microinjection of the dominant negative GTPase mutants N17CDC42Hs, N17Rac or the Rho-specific inhibitor C3-transferase into macrophages greatly attenuated invasin-induced formation of cups. These data suggest that during invasin-triggered phagocytosis β1 integrins activate actin polymerization via CDC42Hs, its effector WASp and the Arp2/3 complex. The contribution of Rac and Rho to phagocytic cup formation also suggests a complex interplay between different Rho GTPases during phagocytosis of pathogens.     

Inhibiting adenosine deaminase modulates the systemic inflammatory response syndrome in endotoxemia and sepsis

By pharmacological manipulation of endogenous adenosine, using chemically distinct methods, we tested the hypothesis that endogenous adenosine tempers proinflammatory cytokine responses and oxyradical-mediated tissue damage during endotoxemia and sepsis. Rats were pretreated with varying doses of pentostatin (PNT; adenosine deaminase inhibitor) or 8-sulfophenyltheophylline (8-SPT; adenosine receptor antagonist) and then received either E. coli endotoxin (lipopolysaccharide; 0.01 or 2.0 mg/kg) or a slurry of cecal matter in 5% dextrose in water (200 mg/kg). Resultant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 were measured in serum and in liver and spleen. Untreated, 2 mg/kg lipopolysaccharide elevated serum TNF-alpha, IL-1beta, and IL-10. PNT dose dependently attenuated, without ablating, the elevation in serum TNF-alpha and IL-1beta and raised liver and spleen IL-10. PNT also attenuated elevation of TNF-alpha in serum, liver, and spleen at 4 and 24 h after sepsis induction, and 8-SPT resulted in higher proinflammatory cytokines. Modulating endogenous adenosine was also effective in exacerbated (8-SPT) or diminished (PNT) tissue peroxidation. Survival from sepsis was also improved when PNT was used as a posttreatment. These data indicate that endogenous adenosine is an important modulatory component of systemic inflammatory response syndromes. These data also indicate that inhibition of adenosine deaminase may be a novel and viable therapeutic approach to managing the systemic inflammatory response syndrome without ablating important physiological functions.     

The mechanism of excitation-contraction coupling in phenylephrine-stimulated human saphenous vein

The human saphenous vein (HSV) is the most widely used graft in coronary artery revascularization procedures and is susceptible to spasm perioperatively. The aim of this study is to elucidate the mechanism(s) of agonist-induced excitation-contraction coupling in this vessel. Isometric contraction experiments were combined with in situ smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) imaging by confocal microscopy of intact undistended HSV segments during activation with phenylephrine (PE; 50 microM). Stimulation with PE produced a sustained contraction. Preincubation with 5 microM nifedipine, a blocker of the L-type voltage-operated Ca(2+) channel, or 50 microM SKF-96365, a blocker of both the voltage- and receptor-operated channels, reduced force generation by 25-30%. Ca(2+) imaging revealed that PE elicited only a transient rise in [Ca(2+)](i), suggesting that Ca(2+) plays only a minor role. However, a requirement for basal Ca(2+) levels was demonstrated when PE contractions could not be maintained in Ca(2+)-free medium. In light of the transient Ca(2+) response, it appears that signals other than Ca(2+) must maintain the tonic contraction elicited by PE, such as those that sensitize the myofilaments to Ca(2+). Application of HA-1077 (a Rho kinase inhibitor) at the peak of the contraction completely abolished the plateau phase of the response, whereas application of genistein (a tyrosine kinase inhibitor) reduced this phase by approximately 50%. The foregoing results suggest that, whereas the transient Ca(2+) signal can contribute to the development of force, maintenance of the plateau phase of the PE contraction in the HSV is the result of myofilament Ca(2+) sensitization by Rho kinase and tyrosine phosphorylation. The elucidation of the mechanisms of excitation-contraction coupling in the HSV may be useful for the development of therapeutic strategies for the alleviation of vein graft spasm.     

Rapid evolution of escape ability in Trinidadian guppies (Poecilia reticulata)

Predators are widely assumed to create selection that shapes the evolution of prey escape abilities. However, this assumption is difficult to test directly due to the challenge of recording both predation and its evolutionary consequences in the wild. We examined these events by studying natural and experimental populations of Trinidadian guppies, Poecilia reticulata, which occur in distinct high-predation and low-predation environments within streams. Importantly, in the last two decades several populations of guppies have been experimentally introduced from one type of predatory environment into the other, allowing measurements of the consequences of change. We used this system to test two hypotheses: First, that changes in predatory environments create phenotypic selection favoring changes in escape ability of guppies, and second, that this selection can result in rapid evolution. For the first test we compared escape ability of wild caught guppies from high- versus low-predation environments by measuring survival rates during staged encounters with a major predator, the pike cichlid Crenicichla alta. We used guppies from three streams, comparing two within-stream pairs of natural populations and three within-stream pairs of an introduced population versus its natural source population. In every comparison, guppies from the high-predation population showed higher survival. These multiple, parallel divergences in guppy survival phenotype suggest that predatory environment does create selection of escape ability. We tested our second hypothesis by rearing guppies in common garden conditions in the laboratory, then repeating the earlier experiments using the F2 generation. As before, each comparison resulted in higher survival of guppies descended from the high-predation populations, demonstrating that population differences in escape ability have a genetic basis. These results also show that escape ability can evolve very rapidly in nature, that is, within 26-36 generations in the introduced populations. Interestingly, we found rapid evolutionary loss of escape ability in populations introduced into low-predation environments, suggesting that steep fitness trade-offs may influence the evolution of escape traits.