Rab11b resides in a vesicular compartment distinct from Rab11a in parietal cells and other epithelial cells

The Rab11 family of small GTPases is composed of three members, Rab11a, Rab11b, and Rab25. While recent work on Rab11a and Rab25 has yielded some insights into their function, Rab11b has received little attention. Therefore, we sought to examine the distribution of endogenous Rab11b in epithelial cells. In rabbit gastric parietal cells, unlike Rab11a, Rab11b did not colocalize or coisolate with H(+)/K(+)-ATPase. In MDCK cells, endogenous Rab11b localized to an apical pericentrisomal region distinct from Rab11a. The microtubule agents nocodazole and taxol dramatically alter Rab11a's localization in the cell, while effects on Rab11b's distribution were less apparent. These results indicate that in contrast to Rab11a, the Rab11b compartment in the apical region is not as dependent upon microtubules. While Rab11a is known to regulate transferrin trafficking in nonpolarized cells and IgA trafficking in polarized cells, Rab11b exhibited little colocalization with either of these cargoes. Thus, while Rab11a and Rab11b share high sequence homology, they appear to reside within distinct vesicle compartments.     

Breakdown of CTL tolerance to self HLA-B*2705 induced by exposure to Chlamydia trachomatis

There is a strong association between seronegative arthritis and HLA B27, but it is still unresolved whether the contribution of B27 to disease pathogenesis is solely as a restriction element for an arthritogenic peptide, or whether B27 itself serves as an autoantigen. This study uses transgenic rats to address the question as to whether exposure to an arthritogenic pathogen can alter tolerance to B27. Unlike their nontransgenic counterparts, B27-transgenic rats are tolerant of B27 immunization using either B27(+) splenocytes or plasmid DNA and do not develop anti-B27 CTL. However, if splenocytes from such immunized animals are exposed to Chlamydia in vitro, CTL are generated that lyse B27(+) targets. No killing was seen with targets transfected with control B7, B14, B40, or B44. This phenomenon was not observed with immunization by nontransgenic splenocytes, or HLA-A2 DNA alone. Using targets expressing mutated B27, we show that the epitope for autoreactive CTL recognition of B27 involves the Lys(70) amino acid residue in the alpha(1) domain of the MHC class I molecule. The generation of CTL with specificity for B27 under these conditions demonstrates that tolerance to B27 can be subverted by CHLAMYDIA: This indicates a dynamic interrelationship between the pathogen and B27, which may have important implications for B27-related spondyloarthropathies triggered by intracellular bacteria.     

Cleavage of the carboxyl tail from the G3 domain of aggrecan but not versican and identification of the amino acids involved in the degradation

Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan.     

Project Grow-2-Gether: a study of the genetic and environmental influences on child eating and obesity.

"Project Grow-2-Gether" is a child nutrition study of same-sex, 3- to 7-year-old monozygotic and dizygotic twin pairs. The study attempts to bridge two bodies of literature that have rarely interfaced with respect to obesity and ingestive behavior: the first being behavioral genetic approaches to obesity-related traits, and the second being developmental approaches focusing on parent-child relationships. The overarching aim of Project Grow-2-Gether is to disentangle genetic from potential home-environmental influences on child eating behavior and body fat. This paper reviews the rationale for Project Grow-2-Gether, its procedures, and core phenotypic measurement battery. A focus of the study is acquisition of controlled food intake measurements obtained in the laboratory, measurement of specific home environmental variables, and multi-method evaluation of parent-child feeding relations. Future directions may involve longitudinal assessment of child growth and molecular analyses for specific genes that influence child eating behavior.     

Identification of an endocrine disrupting agent from corn with mitogenic activity

A mitogenic agent in corncob bedding and fresh corn products disrupts sexual behavior and estrous cyclicity in rats. The mitogenic activity resides in an isomeric mixture of linoleic acid derivatives with a tetrahydrofuran ring and two hydroxyl groups (THF-diols) that include 9, (12)-oxy-10,13-dihydroxystearic acid and 10, (13)-oxy-9,12-dihydroxystearic acid. Synthetic THF-diols stimulated breast cancer cell proliferation in vitro and disrupted the estrous cycle in female rats at oral doses of approximately 0.30 mg/kg body weight/day. Exposure to THF-diols may disrupt endocrine function in experimental animals at doses approximately 200 times lower than classical phytoestrogens, promote proliferation of breast or prostate cancer, and adversely affect human health.     

Inactivation of HIV-1 nucleocapsid protein P7 by pyridinioalkanoyl thioesters. Characterization of reaction products and proposed mechanism of action

The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.     

Exploring the Prevalence of Disrespect and Abuse during Childbirth in Kenya

Background

Poor quality of care including fear of disrespect and abuse (D&A) perpetuated by health workers influences women’s decisions to seek maternity care. Key manifestations of D&A include: physical abuse, non-consented care, non-confidential care, non-dignified care, discrimination, abandonment, and detention in facilities. This paper describes manifestations of D&A experienced in Kenya and measures their prevalence.

Methods

This paper is based on baseline data collected during a before-and-after study designed to measure the effect of a package of interventions to reduce the prevalence of D&A experienced by women during labor and delivery in thirteen Kenyan health facilities. Data were collected through an exit survey of 641 women discharged from postnatal wards. We present percentages of D&A manifestations and odds ratios of its relationship with demographic characteristics using a multivariate fixed effects logistic regression model.

Results

Twenty percent of women reported any form of D&A. Manifestations of D&A includes: non-confidential care (8.5%), non-dignified care (18%), neglect or abandonment (14.3%), Non-consensual care (4.3%) physical abuse (4.2%) and, detainment for non-payment of fees (8.1). Women aged 20-29 years were less likely to experience non-confidential care compared to those under 19; OR: [0.6 95% CI (0.36, 0.90); p=0.017]. Clients with no companion during delivery were less likely to experience inappropriate demands for payment; OR: [0.49 (0.26, 0.95); p=0.037]; while women with higher parities were three times more likely to be detained for lack of payment and five times more likely to be bribed compared to those experiencing there first birth.

Conclusion

One out of five women experienced feeling humiliated during labor and delivery. Six categories of D&A during childbirth in Kenya were reported. Understanding the prevalence of D&A is critical in developing interventions at national, health facility and community levels to address the factors and drivers that influence D&A in facilities and to encourage clients’ future facility utilization.     

Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis

Extracellular-signal regulated kinase (ERK) activation by MEK plays a key role in many of the cellular processes that underlie progressive kidney fibrosis including cell proliferation, apoptosis and transforming growth factor β1-mediated epithelial to mesenchymal transition. We therefore assessed the therapeutic impact of ERK1/2 inhibition using a MEK inhibitor in the rat 5/6 subtotal nephrectomy (SNx) model of kidney fibrosis. There was a twentyfold upregulation in phospho-ERK1/2 expression in the kidney after SNx in Male Wistar rats. Rats undergoing SNx became hypertensive, proteinuric and developed progressive kidney failure with reduced creatinine clearance. Treatment with the MEK inhibitor, CI-1040 abolished phospho- ERK1/2 expression in kidney tissue and prevented phospho-ERK1/2 expression in peripheral lymphocytes during the entire course of therapy. CI-1040 had no impact on creatinine clearance, proteinuria, glomerular and tubular fibrosis, and α-smooth muscle actin expression. However, inhibition of ERK1/2 activation led to significant compensatory upregulation of the MAP kinases, p38 and JNK in kidney tissue. CI-1040 also increased the expression of plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasmin-dependent matrix metalloproteinases. Thus inhibition of ERK1/2 activation has no therapeutic effect on kidney fibrosis in SNx possibly due to increased compensatory activation of the p38 and JNK signalling pathways with subsequent upregulation of PAI-1.     

Discovery of dihydrothieno- and dihydrofuropyrimidines as potent pan Akt inhibitors

Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.