Monitoring the effects of drug treatment in rat models of disease by serum protein analysis

In this review we list from literature investigations on rat serum proteins using electrophoretic techniques in connection with drug testing. From our own research work, we provide annotated two-dimensional maps of rat serum proteins under control and experimental conditions. Emphasis is on species-specific components and on the effects of acute and chronic inflammation. We discuss our project of structural proteomics on rat serum as a minimally invasive approach to pharmacological investigation, and we outline a typical experimental plan for drug testing according to the above guidelines. We then report in detail on the results of our trials of anti-inflammatory drugs on adjuvant arthritis, an animal model of disease resembling in many aspects human rheumatoid arthritis. We demonstrate a correlation between biochemical parameters and therapeutic findings and outline the advantages of the chosen methodological approach, which proved also sensitive in revealing "side effects" of the test drugs. In an appendix we describe our experimental protocol when performing two-dimensional electrophoresis of rat serum.     

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.     

Stage-specific requirement of a mitogen-activated protein kinase by Trypanosoma brucei

In cycling between the mammalian host and the tsetse fly vector, African trypanosomes undergo adaptive differentiation steps that are coupled to growth control. The signaling pathways underlying these cellular processes are largely unknown. Mitogen-activated protein kinases (MAPKs) are known mediators of growth and differentiation in other eukaryotic organisms. To establish the function of a MAPK homologue, TbMAPK2, in T. brucei, a null mutant was constructed. Bloodstream forms of a deltamapk2/deltamapk2 clone were able to grow normally and exhibited no detectable phenotype. When these cells were triggered to differentiate in vitro, however, they developed to the procyclic (fly midgut) form with delayed kinetics and subsequently underwent cell cycle arrest. Introduction of an ectopic copy of the TbMAPK2 gene into the null mutant restored its ability to differentiate and to divide. In contrast, a TbMAPK2 mutant, in which the T190 and Y192 residues of the activating phosphorylation site were replaced by A and F, was unable to restore the growth and differentiation phenotypes. Analysis of the DNA content and the nucleus/kinetoplast configuration of individual cells showed that the null mutant was arrested in all phases of the cell cycle and that 25-30% of the cells had failed to segregate their nucleus and kinetoplast correctly. This implies that cell cycle progression by the procyclic form depends on a constitutive stimulus exerted by the signaling cascade operating through TbMAPK2.     

The alpha 2-adrenergic receptor gene and body fat content and distribution: the HERITAGE Family Study

BACKGROUND: Among adrenergic receptor subtypes that regulate lipid mobilization, the alpha2-adrenergic receptor is involved in the inhibition of fatty acid mobilization from adipose tissue. A C-1291G polymorphism is located in the alpha2-adrenergic receptor gene (ADRA2A) but no association with body fat accumulation has been reported yet. MATERIALS AND METHODS: Body mass index (BMI), fat mass (FAT), percentage body fat (%FAT), trunk-to-extremity skinfold ratio (TER), sum of eight skinfolds (SF8), and abdominal subcutaneous (ASF), visceral (AVF), and total (ATF) fat areas assessed by CT scan have been measured in adult sedentary white (n = 503) and black (n = 276) subjects participating in the HERITAGE Family Study. Association between the C-1291G polymorphism and each phenotype was tested separately in men and women of each race using ANCOVA with the effects of age as covariate in addition to the effects of BMI for TER and of FAT for AVF, ASF, and ATF. RESULTS: The allele frequencies of the ADRA2A C-1291G polymorphism differed between races. No association was observed in white subjects, except for a moderate effect of the polymorphism accounting for less than 1% of the variance in AVF and ATF in women. In black subjects, however, the G-1291 allele was found to be associated with an increase of TER in men (3.8% of variance accounted for by the polymorphism), while in black women it was associated with a decrease in TER (2.9%) and in AVF (2.5%). CONCLUSION: These results suggest a role for the ADRA2A gene in determining the propensity to store fat in the abdominal area, independently of total body fatness.     

Subunit composition and functional properties of G-protein heterotrimers on rat chromaffin granules

Heterotrimeric G-proteins at the plasma membrane serve as switches between heptahelical receptors and intracellular signal cascades. Likewise endomembrane associated G-proteins may transduce signals from intracellular compartments provided they consist of a functional trimer. Using quantitative immunoelectron microscopy we found heterotrimeric G-protein subunits Galpha2, Galpha(q/11), Gbeta2 and Gbeta5 to reside on secretory granules in chromaffin cells of rat adrenal glands.Thus rat chromaffin granules are equipped with functional G-proteins that consist of a specific alpha-, beta- and probably gamma-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Galphao2 (10 nM) to nearly the same extent as by GMppNp (50 microM) whereas GDPbetaS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Galphao2 appears to be the main regulator of vesicular momoamine transporter activity.     

Distinct epithelial responses in experimental colitis: implications for ion uptake and mucosal protection

In the present study, we aimed to investigate enterocyte- and goblet cell-specific functions during the different phases of acute colitis induced with dextran sulfate sodium (DSS). Rats were treated with DSS for 7 days, followed by a 7-day recovery period. Colonic tissue was excised on days 2 (onset of disease), 7 (active disease), and 14 (regenerative phase). Enterocyte functions were studied by the expression of carbonic anhydrases (CAs), sodium/hydrogen exchangers (NHEs) and intestinal fatty acid-binding protein (iFABP) and by alkaline phosphatase (AP) activity. The expression and secretion of the mucin Muc2 and trefoil factor family peptide-3 (TFF3) were used as parameters for goblet cell function. DSS induced a downregulation of the CAs, NHEs, and iFABP in some normal-appearing surface enterocytes and in most of the flattened-surface enterocytes during disease onset and active disease. During the regenerative phase most enterocytes expressed these genes again. Quantitative analysis revealed a significant decrease in CAs, NHEs, and iFABP expression levels during onset and active disease. During the regenerative phase, the expression levels of the CAs were restored, whereas the expression levels of the NHEs and iFABP remained decreased. In contrast, enterocyte-specific AP activity was maintained in normal and flattened enterocytes during DSS-induced colitis. Goblet cells continued to express MUC2 and TFF3 during and after DSS treatment. Moreover, Muc2 and TFF3 expression and secretion levels were maintained or even increased during each of the DSS-induced disease phases. In conclusion, DSS-induced colitis was associated with decreased expression of CAs, NHEs, and iFABP. The loss of these genes possibly accounts for some of the pathology seen in colitis. The maintenance or upregulation of Muc2 and TFF3 synthesis and secretion levels implies that goblet cells at least maintain their epithelial defense and repair capacity during acute inflammation induced by DSS.     

A maternal Smad protein regulates early embryonic apoptosis in Xenopus laevis

We identified cDNAs encoding the Xenopus Smad proteins most closely related to mammalian Smad8, and we present a functional analysis of this activity (also referred to recently as xSmad11). Misexpression experiments indicate that xSmad8(11) regulates pathways distinct from those regulated by the closely related xSmad1. Embryos that develop from eggs depleted of xSmad8(11) mRNA fail to gastrulate; instead, at the time of gastrulation, they initiate a widespread program of apoptosis, via a CPP32/caspase 3 pathway. Embryos that avoid this fate display gastrulation defects. Activation of apoptosis is rescued by expression of xSmad8(11) but not xSmad1. Our results demonstrate an embryonic requirement for Smad8(11) activity and show that a maternally derived Smad signaling pathway is required for gastrulation and for mediating a cell survival program during early embryogenesis. We suggest that xSmad8(11) functions as part of a maternally derived mechanism shown previously by others to monitor Xenopus early embryonic cell cycles.     

Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA

The mild phenotype associated with targeted disruption of the mouse OGG1 and NTH1 genes has been attributed to the existence of back-up activities and/or alternative pathways for the removal of oxidised DNA bases. We have characterised two new genes in human cells that encode DNA glycosylases, homologous to the bacterial Fpg (MutM)/Nei class of enzymes, capable of removing lesions that are substrates for both hOGG1 and hNTH1. One gene, designated HFPG1, showed ubiquitous expression in all tissues examined whereas the second gene, HFPG2, was only expressed at detectable levels in the thymus and testis. Transient transfections of HeLa cells with fusions of the cDNAs to EGFP revealed intracellular sorting to the nucleus with accumulation in the nucleoli for hFPG1, while hFPG2 co-localised with the 30 kDa subunit of RPA. hFPG1 was purified and shown to act on DNA substrates containing 8-oxoguanine, 5-hydroxycytosine and abasic sites. Removal of 8-oxoguanine, but not cleavage at abasic sites, was opposite base-dependent, with 8-oxoG:C being the preferred substrate and negligible activity towards 8-oxoG:A. It thus appears that hFPG1 has properties similar to mammalian OGG1 in preventing mutations arising from misincorporation of A across 8-oxoG and could function as a back-up repair activity for OGG1 in ogg1(-/-) mice.     

A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector

The stimulatory effect of VIP on intracellular calcium concentration ([Ca(2+)](i)) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC(1), VPAC(2), chimeric VPAC(1)/VPAC(2), or mutated receptors. The VIP-induced [Ca(2+)](i) increase was linearly correlated with receptor density and was higher in cells expressing VPAC(1) receptors than in cells expressing a similar VPAC(2) receptor density. The study was performed to establish the receptor sequence responsible for that difference. VPAC(1)/VPAC(2) chimeric receptors were first used for a broad positioning: those having the third intracellular loop (IC(3)) of the VPAC(1) or of the VPAC(2) receptor behaved, in that respect, phenotypically like VPAC(1) and VPAC(2) receptor, respectively. Replacement in the VPAC(2) receptor of the sequence 315-318 (VGGN) within the IC(3) by its VPAC(1) receptor counterpart 328-331 (IRKS) and the introduction of VGGN in state of IRKS in VPAC(1) was sufficient to mimic the VPAC(1) and VPAC(2) receptor characteristics, respectively. Thus, a small sequence in the IC(3) of the VPAC(1) receptor, probably through interaction with G(alphai) and G(alphaq) proteins, is responsible for the efficient agonist-stimulated [Ca(2+)](i) increase.