Fine-tuning function: correlation of hinge domain interactions with functional distinctions between LacI and PurR

LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function.     

Effects of scrotal heating on sperm surface protein PH-20 expression in sheep

Sperm surface protein PH-20 expression was studied during spermatogenesis in pubertal and adult sheep, using molecular and histological methods. The effects of 24 hr of insulation raising scrotal temperatures to 39 degrees C on PH-20 expression in ejaculated sheep sperm were also determined. A 282 nt cDNA fragment of ovine PH-20 was identified in total RNA extracts of sheep testes, which exhibited 76% identity at the nucleotide level with the equivalent region of the human sequence. Ovine PH-20 mRNA and immunoreactivity were identified only in adult ram testis and not in peri-pubertal ram testis tubules lacking round spermatids, nor in adult sheep brain, pituitary, heart, spleen, lung, liver, kidney, epididymis, or ovary. Ovine PH-20 protein was distributed predominantly on the postacrosomal membrane and was also present on the anterior membrane of the sperm head in fresh, unheated sheep semen. Scrotal heating caused a significant, transient decrease in the percentage of PH-20 immunoreactive sperm, but did not change the pattern of PH-20 staining on the sperm head. The results strongly suggest that ovine PH-20 is postmeiotically expressed in haploid germ cells in sheep testis and is arrayed on the membrane of the mature ovine spermatozoon. Scrotal heating appears to have few effects on PH-20 expression and distribution on ejaculated sperm.     

Gene expression in the normal adult human kidney assessed by complementary DNA microarray

The kidney is a highly specialized organ with a complex, stereotyped architecture and a great diversity of functions and cell types. Because the microscopic organization of the nephron, the functional unit of the kidney, has a consistent relationship to the macroscopic anatomy of the kidney, knowledge of the characteristic patterns of gene expression in different compartments of the kidney could provide insight into the functions and functional organization of the normal nephron. We studied gene expression in dissected renal lobes of five adult human kidneys using cDNA microarrays representing approximately 30,000 different human genes. Total RNA was isolated from sections of the inner and outer cortex, inner and outer medulla, papillary tips, and renal pelvis and from glomeruli isolated by sieving. The results revealed unique and highly distinctive patterns of gene expression for glomeruli, cortex, medulla, papillary tips, and pelvic samples. Immunohistochemical staining using selected antisera confirmed differential expression of several cognate proteins and provided histological localization of expression within the nephron. The distinctive patterns of gene expression in discrete portions of the kidney may serve as a resource for further understanding of renal physiology and the molecular and cellular organization of the nephron.     

ERK signaling pathway is differentially involved in erythroid differentiation of K562 cells depending on time and the inducing agent

K562 cells can be induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, butyrate, cisplatin and ara-C. Differential signaling through MAP kinases has been suggested to be involved in this differentiation process. We have investigated the involvement of ERK activation/inhibition in hemin-, butyrate-, cisplatin- and ara-C-induced erythroid differentiation using the K562 cell line. ERK activity decreased for 2-4h after administration of either inducing agent. ERK was then activated by hemin and cisplatin, while ERK phosphorylation remained decreased during incubation with butyrate and ara-C. There was no activation of JNK or p38. The MEK-1 inhibitors UO126 or PD98059 induced erythroid differentiation in K562 cells and acted additively with butyrate. Inhibition of MEK-1 reduced the hemoglobin accumulation by hemin and cisplatin; erythroid differentiation by ara-C was unchanged. The results suggest that inhibition of signaling through ERK in K562 cells may be needed to enter the erythroid differentiation process, while after initiation both activation and inhibition of signaling through ERK enhance erythroid differentiation, which, however, is dependent on the inducing compound.     

Sensory integration in the hydrodynamic world of rainbow trout

Water movements, of both abiotic and biotic origin, provide a wealth of information for fishes. They detect these water movements by arrays of hydrodynamic sensors located on the surface of the body as superficial neuromasts and embedded in subdermal lateral line canals. Recently, the anatomical dichotomy between superficial and canal neuromasts has been matched by demonstrations of a corresponding functional dichotomy. Superficial neuromasts are sensitive to water flows over the surface of the fish and are the sub-modality that participates in orientation to water currents, a behaviour known as rheotaxis. The canal neuromasts are sensitive to water vibration and it is this sub-modality that determines the localization of artificial prey. Recently, however, it has been shown that the complex behaviour of natural prey capture in the dark requires input from both lateral line sensory submodalities and here we show that the ability of trout to hold station behind a stationary object in fast flowing water also requires integration of information from both sub-modalities.     

Studies investigating pretransplant donor-specific blood transfusion,rapamycin, and the CD154-specific antibody IDEC-131 in a nonhuman primate model of skin allotransplantation

Anti-CD154 variably prolongs allograft survival in nonhuman primates. Rodent studies suggest that adding pretransplant donor-specific transfusion (DST) and/or rapamycin to anti-CD154 improves survival. The CD154-specific Ab IDEC-131 was tested alone and in combination with rapamycin for its ability to inhibit rhesus MLRs. The ability of the Ab to block endothelial activation was also assessed. IDEC-131 was then tested alone and in combination with DST and/or rapamycin for its ability to prevent rejection of full-thickness, MHC-mismatched rhesus skin allografts. Animals were monitored for donor-specific hyporesponsiveness by MLR and alloantibody determination. IDEC-131 modestly inhibited rhesus MLRs and inhibited CD154-dependent endothelial cell activation. Rapamycin combined with IDEC-131 additively inhibited MLRs. IDEC-131 modestly prolonged allograft survival when compared with no treatment, rapamycin alone, or DST plus rapamycin. Adding DST to IDEC-131 did not prolong survival beyond IDEC-131 alone. IDEC-131 plus rapamycin was effective in prolonging graft survival, although animals had episodes of acute rejection before graft demise. Therapy with IDEC-131, rapamycin, and DST induced long-term allograft survival without intermittent acute rejection. However, no evidence for MLR inhibition was seen, and most animals eventually developed alloantibody. All animals ultimately rejected their grafts after drug withdrawal. IDEC-131 modestly prolongs rhesus skin allograft survival. Rapamycin and rapamycin plus DST improves the efficacy of IDEC-131 in prolonging allograft survival. IDEC-131, rapamycin, and DST are a promising combination for clinical evaluation in allotransplantation.     

Slipped (CTG).(CAG) repeats of the myotonic dystrophy locus: surface probing with anti-DNA antibodies

At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x相似文献   

mRNA localization in polarized intestinal epithelial cells

An important feature of enterocyte maturation is the asymmetrical distribution of cellular functions including protein localization. mRNA sorting is one mechanism for establishment and maintenance of this process in other systems, and we have previously demonstrated differential localization of mRNAs in human enterocytes. To study regulation of mRNA sorting, we established a model in polarized Caco-2 cells. Proxy cDNA constructs containing beta-galactosidase (beta-gal)/green fluorescence protein (GFP) and the 3'-untranslated region (3'-UTR) of either human sucrase-isomaltase or villin were transfected transiently or stably. A control construct contained poly-A sequence in place of 3'-UTR. Expression of GFP was observed by confocal microscopy; intracellular location of the construct mRNA was imaged by in situ hybridization. The sucrase-isomaltase mRNA proxy localized to an apical position in Caco-2 cells as in native enterocytes; the villin mRNA proxy did not show significant localization. The control construct was not localized and was found diffusely throughout the cell. Proxy GFP proteins tended to localize with their mRNA proxies, but with less precision. This study establishes a valuable model for the investigation of mRNA localization in intestinal epithelial cells. Mechanisms controlling asymmetrical distribution of intestinal mRNAs can be now be elucidated.     

Mapping of the laminin-binding site of the N-terminal agrin domain (NtA)

Agrin is a key organizer of acetylcholine receptor (AChR) clustering at the neuromuscular junction. The binding of agrin to laminin is required for its localization to synaptic basal lamina and other basement membranes. The high-affinity interaction with the coiled-coil domain of laminin is mediated by the N-terminal domain of agrin. We have adopted a structurally guided site-directed mutagenesis approach to map the laminin-binding site of NtA. Mutations of L117 and V124 in the C-terminal helix 3 showed that they are crucial for binding. Both residues are located in helix 3 and face the groove between the beta-barrel and the C-terminal helical segment of NtA. Remarkably, the distance between both residues matches a heptad repeat distance of two aliphatic residues which are solvent exposed in the coiled-coil domain of laminin. A lower but significant contribution originates from R43 and a charged cluster (E23, E24 and R40) at the open face of the beta-barrel structure. We propose that surface-exposed, conserved residues of the laminin gamma1 chain interact with NtA via hydrophobic and ionic interactions.     

Intracellular signaling molecules involved in an inhibitory factor-induced decrease in fetal-type AChR expression

The innervation-induced down-regulation of fetal-type acetylcholine receptor (AChR) expression in developing muscle fibers has largely been attributed to nerve-evoked muscle activity; however, there is increasing evidence that a neural trophic factor also contributes to this receptor down-regulation. Previous studies from this laboratory have shown that neural extracts contain a factor which decreases fetal-type AChR expression in skeletal muscle cell lines and therefore may account for the proposed inhibitory neurotrophic influence. The current study investigated possible intracellular signaling molecules involved in this receptor down-regulation and demonstrated that activation of protein kinase C and p70(S6k) appeared to be important in receptor down-regulation. Decreases in AChR density were independent of myogenin. In addition, the receptor down-regulation was independent of neuregulin, which also induces p70(S6k) activity. These studies demonstrate that neural extracts contain an inhibitory factor which can down-regulate fetal-type AChR expression independently of nerve-evoked muscle activity through intracellular signaling molecules which are known to regulate AChR expression.