G proteins and small GTPases: distant relatives keep in touch.
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Actins↗
Biomedical subjects
A Hall
Publications and source records attributed to A Hall.
Activation of G1 progression, JNK mitogen-activated protein kinase, and actin filament assembly by the exchange factor FGD1.
Cdc42 has been shown to control bifurcating pathways leading to filopodia formation/G1 cell cycle progression and to JNK mitogen-activated protein kinase activation. To dissect these pathways further, the cellular effects induced by a Cdc42 guanine nucleotide exchange factor, FGD1, have been examined. All exchange factors acting on the Rho GTPase family have juxtaposed Dbl homology (DH) and pleckstrin homology (PH) domains. We report here that FGD1 triggers G1 cell cycle progression and filopodia formation in Swiss 3T3 fibroblasts as well as JNK mitogen-activated protein kinase activation in COS cell transfection assays. FGD1-induced filopodia formation is Cdc42-dependent, and both the DH and PH domains are essential. Although expression of the FGD1 DH domain alone does not activate Cdc42 and induce filopodia, it does trigger both the JNK cascade in COS cells and G1 progression in quiescent Swiss 3T3 cells. We conclude that FGD1 can trigger G1 progression independently of actin polymerization or integrin adhesion complex assembly. Furthermore, since FGD1 activates JNK and G1 progression in a Cdc42-independent manner, it must have additional, as yet unidentified, targets.
3T3 Cells↗
A new member of the Rho family, Rnd1, promotes disassembly of actin filament structures and loss of cell adhesion.
Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell-substrate adhesion leading to cell rounding (hence Rnd for "round"). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.
3T3 Cells↗
Structural basis for different inhibitory specificities of human cystatins C and D.
Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S. To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems. In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold. Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity. The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured. By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H. These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively). Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C. Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency. Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D.
Amino Acid Sequence↗
A new rac target POSH is an SH3-containing scaffold protein involved in the JNK and NF-kappaB signalling pathways.
The Rho, Rac and Cdc42 GTPases coordinately regulate the organization of the actin cytoskeleton and the JNK MAP kinase pathway. Mutational analysis of Rac has previously shown that these two activities are mediated by distinct cellular targets, though their identity is not known. Two Rac targets, p65(PAK) and MLK, are ser/thr kinases that have been reported to be capable of activating the JNK pathway. We present evidence that neither is the Rac target mediating JNK activation in Cos-1 cells. We have used yeast two-hybrid selection and identified a new target of Rac, POSH. This protein consists of four SH3 domains and ectopic expression leads to the activation of the JNK pathway and to nuclear translocation of NF-kappaB. When overexpressed in fibroblasts, POSH is a strong inducer of apoptosis. We propose that POSH acts as a scaffold protein and contributes to Rac-induced signal transduction pathways leading to diverse gene transcriptional changes.
3T3 Cells↗
PRK1 is targeted to endosomes by the small GTPase, RhoB.
RhoB has been shown to be an endosomal GTPase both by immunocytochemistry and electron microscopy, however, its role in endocytosis is unknown. Elucidation of the cellular roles of other members of this superfamily of signaling proteins has come with the identification of their downstream partners. We show here that the recently isolated serine/threonine kinase PRK1 is targeted to the endosomal compartment by RhoB. This is established both through immunofluorescence and cell fractionation. PRK1 is shown to interact with activated RhoB in cells and is localized to endosomes through its Rho-binding HR1 domain. Translocation of PRK1 to the endosomal compartment by RhoB is accompanied by a shift in the electrophoretic mobility of the kinase indicative of an accompanying activation.
3T3 Cells↗
Polymerase chain reaction as marker of infectivity in people with hepatitis C. Summary vertical transmission rates may be misleading.
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Biomarkers↗
Rho GTPases and the actin cytoskeleton.
The actin cytoskeleton mediates a variety of essential biological functions in all eukaryotic cells. In addition to providing a structural framework around which cell shape and polarity are defined, its dynamic properties provide the driving force for cells to move and to divide. Understanding the biochemical mechanisms that control the organization of actin is thus a major goal of contemporary cell biology, with implications for health and disease. Members of the Rho family of small guanosine triphosphatases have emerged as key regulators of the actin cytoskeleton, and furthermore, through their interaction with multiple target proteins, they ensure coordinated control of other cellular activities such as gene transcription and adhesion.
Actins↗
The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.
The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.
14-3-3 Proteins↗
Physiological stability of preterm infants during magnetic resonance imaging.
AIM: This study looked for evidence of physiological disturbance in preterm infants undergoing magnetic resonance imaging (MRI). METHODS: Intensive care was continued, as appropriate, throughout scanning in each infant. The heart rate, oxygen saturation (SaO2), temperature and mean arterial blood pressure (BP) was monitored during MRI in preterm infants, median gestational age at birth 27 (range 23-32) weeks and median postnatal age at initial MRI, 3 days (range 1-42). The acoustic noise level during imaging was also measured. RESULTS: 2087 min of data were obtained from 39 examinations in 23 infants. The median heart rate was 159 and no bradycardia < 100 or tachycardia > 200 bpm occurred. Although 42 episodes of desaturation < 90% were detected only three were < 80, and these occurred in one infant due to endotracheal tube blockage. The median axillary temperature was 36.9 degrees C (range 35.7-37.8) and median BP (n = 6) was 37 mmHg (24-48). The ambient noise level in the MR system during scanning was 67-72 dBA. CONCLUSION: In preterm infants who required intensive care during scanning, MRI could be performed without major physiological instability.
Blood Pressure↗
Regulation of B cell apoptosis during the cell cycle.
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Apoptosis↗
We have a cancer vaccine--why don't we use it?
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Cancer Vaccines↗
Nosocomial infection in developing countries: time to learn.
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Africa↗
Epidemiology of soil-transmitted nematode infections in Ha Nam Province, Vietnam.
Baseline epidemiological data are presented from a parasitological survey conducted in Thuy Loi commune, Ha Nam province, Vietnam; a farming community where night soil is routinely used as fertilizer for crops. 177 households were visited and 543 individuals (aged 1-88 years) recruited to the study. Helminth infection intensity was assessed by Kato-Katz to determine the density of parasite eggs per gram of stool (epg). Ascaris lumbricoides, Trichuris trichiura and hookworm infections were the only species detected. 83% of individuals were infected with A. lumbricoides (mean epg = 11,971), 94% with T. trichiura (mean epg = 793) and 59% with hookworm (mean epg = 302). Age-dependent patterns of infection prevalence and intensity were similar for A. lumbricoides and T. trichiura, but markedly different for hookworm infection. Similarly, age-dependency in the k-values for the three infections was due to covariance with the respective mean intensities with age rather than to independent age effects, with similar patterns for A. lumbricoides and T. trichiura, and a different pattern for hookworm. Three major conclusions can be drawn from the multiple-species analyses: There is positive interaction between A. lumbricoides and T. trichiura infections; high-intensity A. lumbricoides infections are significantly associated with high-intensity T. trichiura infections; and there is positive interaction between these two species such that infection intensity of A. lumbricoides is higher in individuals with concurrent T. trichiura infection than in individuals without and vice versa.
Adolescent↗
Astrovirus infection in association with acute, persistent and nosocomial diarrhea in Bangladesh.
BACKGROUND: Diarrhea is an important public health concern in developing countries such as Bangladesh. Diarrhea in children that persists for 14 days or more occurs in 7% of patients in Bangladesh and frequently results in death. Astrovirus has been demonstrated as a cause of acute and nosocomial diarrhea and can be excreted for prolonged periods, yet its importance as a cause of diarrhea among children in a developing country like Bangladesh has not been investigated. METHODS: We tested 629 stool specimens from patients with acute diarrhea, 153 from patients with persistent diarrhea, 175 specimens from 76 patients hospitalized for diarrhea who were sampled repeatedly to detect nosocomial infection and 428 from nonhospitalized healthy children (controls). All children enrolled in the study were <5 years of age. Astrovirus was detected by enzyme immunoassay and other enteropathogens were detected by standard techniques. RESULTS: The detection of astrovirus increased significantly with the duration of diarrhea. Astrovirus was found in 23 (15%) specimens from patients with persistent diarrhea, 26 (4%) patients with acute diarrhea, but only 8 (2%) healthy controls. This trend remained when we limited our analysis to infants <12 months of age and to episodes in which astrovirus was the sole pathogen. Among patients with nosocomial diarrhea, 16% of postadmission specimens were positive for astrovirus when the admission specimen was negative. CONCLUSION: The observation that astrovirus is detected more frequently with diarrhea of increasing duration suggests the need for further studies to determine whether astrovirus plays a causative role in persistent diarrhea or is a secondary agent.
Acute Disease↗
Selectivity of ferric enterobactin binding and cooperativity of transport in gram-negative bacteria.
The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their 59Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (Kd approximately 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K3MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (Kd </= 100 nM) and transported it at comparable rates (>/=50 pmol/min/10(9) cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.
Bacterial Outer Membrane Proteins↗
Identification and localization of extracellular Ca(2+)-sensing receptor in rat intestine.
The extracellular calcium (Ca2+o)-sensing receptor (CaR) plays vital roles in Ca2+o homeostasis, but no data are available on its expression in small and large intestine. Polymerase chain reaction products amplified from reverse-transcribed duodenal RNA using CaR-specific primers showed > 99% homology with the rat kidney CaR. Northern analysis with a CaR-specific cRNA probe demonstrated 4.1- and 7.5-kb transcripts in all intestinal segments. Immunohistochemistry with CaR-specific antisera showed clear basal staining of epithelial cells of small intestinal villi and crypts and modest apical staining of the former, whereas there was both basal and apical staining of colonic crypt epithelial cells. In situ hybridization and immunohistochemistry also demonstrated CaR expression in Auerbach's myenteric plexus of small and large intestines and in the submucosa in the region of Meissner's plexus. Our results reveal CaR expression in several cell types of small and large intestine, in which it may modulate absorptive and/or secretomotor functions.
Animals↗