Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Role of the host cell's unfolded protein response in arenavirus infection.

Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.

Breast cancer 1 (BrCa1) may be behind decreased lipogenesis in adipose tissue from obese subjects.

CONTEXT Expression and activity of the main lipogenic enzymes is paradoxically decreased in obesity, but the mechanisms behind these findings are poorly known. Breast Cancer 1 (BrCa1) interacts with acetyl-CoA carboxylase (ACC) reducing the rate of fatty acid biosynthesis. In this study, we aimed to evaluate BrCa1 in human adipose tissue according to obesity and insulin resistance, and in vitro cultured adipocytes. RESEARCH DESIGN AND METHODS BrCa1 gene expression, total and phosphorylated (P-) BrCa1, and ACC were analyzed in adipose tissue samples obtained from a total sample of 133 subjects. BrCa1 expression was also evaluated during in vitro differentiation of human adipocytes and 3T3-L1 cells. RESULTS BrCa1 gene expression was significantly up-regulated in both omental (OM; 1.36-fold, p = 0.002) and subcutaneous (SC; 1.49-fold, p = 0.001) adipose tissue from obese subjects. In parallel with increased BrCa1 mRNA, P-ACC was also up-regulated in SC (p = 0.007) as well as in OM (p = 0.010) fat from obese subjects. Consistent with its role limiting fatty acid biosynthesis, both BrCa1 mRNA (3.5-fold, p<0.0001) and protein (1.2-fold, p = 0.001) were increased in pre-adipocytes, and decreased during in vitro adipogenesis, while P-ACC decreased during differentiation of human adipocytes (p = 0.005) allowing lipid biosynthesis. Interestingly, BrCa1 gene expression in mature adipocytes was restored by inflammatory stimuli (macrophage conditioned medium), whereas lipogenic genes significantly decreased. CONCLUSIONS The specular findings of BrCa1 and lipogenic enzymes in adipose tissue and adipocytes reported here suggest that BrCa1 might help to control fatty acid biosynthesis in adipocytes and adipose tissue from obese subjects.

Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate, chloride and organic-anion transporters.

A protein from Arabidopsis thaliana (L.) Heynh. showing homology to animal proteins of the NaPi-1 family, involved in the transport of inorganic phosphate, chloride, glutamate and sialic acid, has been characterized. This protein, named ANTR2 (for anion transporters) was shown by chloroplast subfractionation to be localized to the plastid inner envelope in both A. thaliana and Spinacia oleracea (L.). Immunolocalization revealed that ANTR2 was expressed in the leaf mesophyll cells as well as in the developing embryo at the upturned-U stage. Five additional homologues of ANTR2 are found in the Arabidopsis genome, of which one was shown by green fluorescent protein (GFP) fusion to be also located in the chloroplast. All ANTR proteins share homology to the animal NaPi-1 family, as well as to other organic-anion transporters that are members of the Anion:Cation Symporter (ACS) family, and share the main features of transporters from this family, including the presence of 12 putative transmembrane domains and of a 7-amino acid motif in the fourth putative transmembrane domain. ANTR2 thus represent a novel protein of the plastid inner envelope that is likely to be involved in anion transport.

Profile of circulating levels of IL-1Ra, CXCL10/IP-10, CCL4/MIP-1β and CCL2/MCP-1 in dengue fever and parvovirosis

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1β, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.

Human bocavirus 1 and 3 infection in children with acute gastroenteritis in Brazil

To determine the positivity rate of human bocavirus (HBoV) 1 and 3 among children who presented with acute gastroenteritis symptoms during the period of 1994-2004 in the Central-West Region of Brazil, 762 faecal samples were tested using polymerase chain reaction (PCR) for the detection of HBoV DNA. Primers for a segment of the non-structural viral protein 1 (NS1) gene of HBoV-1 and HBoV-3 were used. Twelve HBoV-positive samples were further characterised via genomic sequencing and phylogenetic analysis. Of the samples tested, 5.8% (n = 44) were positive for HBoV-1 or HBoV-3 and co-infection was observed in 14 (31.8%) of the 44 HBoV-positive samples. Nine of the 14 samples were also positive for Rotavirus A and five were positive for Aichi virus. The genomic sequencing of the NS1 partial sequence of 12 HBoV-samples showed that 11 samples were characterised as HBoV-1 and that one was characterised as HBoV-3. The phylogenetic analysis showed that the HBoV-1 samples had a high sequence homology to others previously identified in China, Sweden and Brazil. This is the first study conducted in the Central-West Region of Brazil to detect HBoV-1 and HBoV-3 in faecal samples from children with acute gastroenteritis. Further studies are required to define the role of HBoVs as aetiological agents of gastroenteritis.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Association of the solute carrier family 11 member 1 gene polymorphisms with susceptibility to leprosy in a Brazilian sample

Natural resistance-associated macrophage protein 1/solute carrier family 11 member 1 gene (Nramp1/Slc11a1) is a gene that controls the susceptibility of inbred mice to intracellular pathogens. Polymorphisms in the human Slc11a1/Nramp1 gene have been associated with host susceptibility to leprosy. This study has evaluated nine polymorphisms of the Slc11a1/Nramp1 gene [(GT)n, 274C/T, 469+14G/C, 577-18G/A, 823C/T, 1029 C/T, 1465-85G/A, 1703G/A, and 1729+55del4] in 86 leprosy patients (67 and 19 patients had the multibacillary and the paucibacillary clinical forms of the disease, respectively), and 239 healthy controls matched by age, gender, and ethnicity. The frequency of allele 2 of the (GT)n polymorphism was higher in leprosy patients [p = 0.04, odds ratio (OR) = 1.49], whereas the frequency of allele 3 was higher in the control group (p = 0.03; OR = 0.66). Patients carrying the 274T allele (p = 0.04; OR = 1.49) and TT homozygosis (p = 0.02; OR = 2.46), such as the 469+14C allele (p = 0.03; OR = 1.53) of the 274C/T and 469+14G/C polymorphisms, respectively, were more frequent in the leprosy group. The leprosy and control groups had similar frequency of the 577-18G/A, 823C/T, 1029C/T, 1465-85G/A, 1703G/A, and 1729+55del4 polymorphisms. The 274C/T polymorphism in exon 3 and the 469+14G/C polymorphism in intron 4 were associated with susceptibility to leprosy, while the allele 2 and 3 of the (GT)n polymorphism in the promoter region were associated with susceptibility and protection to leprosy, respectively.

High prevalence of anti-apolipoprotein/A-1 autoantibodies in maintenance hemodialysis and association with dialysis vintage.

Autoantibodies to apolipoprotein/A-1 (anti-ApoA-1 IgG) have pro-atherogenic properties in patients at high cardiovascular risk, but its prevalence in patients with end-stage kidney disease is unknown. The aims of this single-center, cross-sectional study were to assess the prevalence of anti-ApoA-1 antibodies in patients on maintenance hemodialysis (MHD), and to examine its correlation with inflammatory biomarkers related to atherosclerotic plaque vulnerability and dialysis vintage. To this purpose, anti-ApoA-1 IgG levels and the concentrations of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), metalloproteinase-9 (MMP-9), tumor necrosis factor-α, and C-reactive protein (CRP) were assessed in the sera of 66 MHD patients (mean age: 68 ± 14 years, 36% women, 32% diabetics). Anti-ApoA-1 IgG positivity (defined as a blood value ≥ 97.5(th) percentile of the normal distribution as assessed in healthy blood donors) was 20%. Circulating levels of anti-ApoA-1 IgG correlated positively with dialysis vintage, but not with cardiovascular risk factors or previous cardiovascular events; no significant correlations were found between the anti-ApoA1 IgG levels and circulating levels of IL-6, IL-8, MCP-1, MMP-9, CRP, or low-density lipoprotein-cholesterol. In multivariable linear regression, adjusted for age and sex, only dialysis vintage remained positively and independently associated with anti-ApoA-1 titers (β = 0.05, 95% CI: 0.006; 0.28, P = 0.049). In conclusion, the prevalence of anti-ApoA-1 IgG is raised in the MHD-population, and positively associated with dialysis vintage, a major determinant of cardiovascular outcome. Whether antiApoA-1 antibodies play a role in the pathophysiology of accelerated atherosclerosis in the MHD-population merits further study.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.

VARIABILITY OF PHENOTYPIC, PROTEOMIC AND TRANSCRIPTOMIC EXPRESSION OF STAPHYLOCOCCUS AUREUS SURFACE ADHESINS

Staphylococcus aureus is a highly successful pathogen responsible of a wide variety of diseases, from minor skin infection to life-threatening sepsis or infective endocarditis, as well as food poisoning and toxic shock syndrome. This heterogeneity of infections and the ability of S. aureus to develop antibiotic-resistance to virtually any available drugs reflect its extraordinary capacity to adapt and survive in a great variety of environments. The pathogenesis of S. aureus infection involves a wide range of cell wall-associated adhesins and extracellular toxins that promote host colonization and invasion. In addition, S. aureus is extremely well equipped with regulatory systems that sense environmental conditions and respond by fine tuning the expression of metabolic and virulence determinants. Surface adhesins referred to MSCRAMMs - for Microbial Surface Component Recognizing Adherence Matrix Molecules - mediate binding to the host extracellular matrix or serum components, including fibrinogen, fibronectin, collagen and elastin, and promote tissue colonization and invasion. Major MSCRAMMs include a family of surface-attached proteins covalently bound to the cell wall peptidoglycan via a conserved LPXTG motif. Genomic analyses indicate that S. aureus contain up to 22 LPXTG surface proteins, which could potentially act individually or in synergy to promote infection. In the first part of this study we determined the range of adherence phenotypes to fibrinogen and fibronectin among 30 carriage isolates of S. aureus and compared it to the adherence phenotypes of 30 infective endocarditis and 30 blood culture isolates. Overall there were great variations in in vitro adherence, but no differences were observed between carriage and infection strains. We further determined the relation between in vitro adherence and in vivo infectivity in a rat model of experimental endocarditis, using 4 isolates that displayed either extremely low or high adherence phenotypes. Unexpectedly, no differences were observed between the in vivo infectivity of isolates that were poorly and highly adherent in vitro. We concluded that the natural variability of in vitro adherence to fibrinogen and fibronectin did not correlate with in vivo infectivity, and thus that pathogenic differences between various strains might only be expressed in in vivo conditions, but not in vitro. Therefore, considering the importance of adhesins expression for infection, direct measurement of those adhesins present on the bacterial surface were made by proteomic approach. 5 In the second series of experiments we assessed the physical presence of the LPXTG species at the staphylococcal surface, as measured at various time points during growth in different culture media. S. aureus Newman was grown in either tryptic soy broth (TSB) or in Roswell Park Memorial Institute (RPMI) culture medium, and samples were removed from early exponential growth phase to late stationary phase. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa) and clumping factor A (ClfA). Peptides of surface proteins were recovered by "trypsin-shaving" of live bacteria, and semi-quantitative proteomic analysis was performed by tandem liquid-chromatography and mass-spectrometry (LC-MS). We also determined in parallel the mRNA expression by microarrays analysis, as well as the phenotypic adherence of the bacteria to fibrinogen in vitro. The surface proteome was highly complex and contained numerous proteins theoretically not belonging to the bacterial envelope, including ribosomal proteins and metabolic enzymes. Sixteen of the 21 known LPXTG species were detected, but were differentially expressed. As expected, 9 known agr-regulated proteins (e.g. including Spa, FnBPA, ClfA, IsdA, IsdB, SasH, SasD, SasG and FmtB) increased up to the late exponential growth phase, and were abrogated in agr-negative mutants. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr negative mutant, while all other LPXTG proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in in vitro fibrinogen adherence tests during late growth (24h), whereas it remained poorly detected by proteomics. Differential expression was also detected in iron-rich TSB versus iron-poor RPMI. Proteins from the iron-regulated surface determinant (isd) system, including IsdA, IsdB and IsdH were barely expressed in iron-rich TSB, whereas they increased their expression by >10 time in iron-poor RPMI. We conclude that semi-quantitative proteomic analysis of specific protein species is feasible in S. aureus and that proteomic, transcriptomic and adherence phenotypes demonstrated differential profiles in S. aureus. Furthermore, peptide signatures released by trypsin shaving suggested differential protein domain exposures in various environments, which might be relevant for antiadhesins vaccines. A comprehensive understanding of the S. aureus physiology should integrate all these approaches.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogeneous disease . The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs 2-5). We evaluated the role of IBi in beta-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma beta-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IBi protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on i49 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (559N) that segregated with diabetes. In vitro, this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of 3-cell function.

Elevated inflammatory markers combined with positive pneumococcal urinary antigen are a good predictor of pneumococcal community-acquired pneumonia in children.

BACKGROUND: Our objective was to evaluate procalcitonin (PCT) and C-reactive protein (CRP) as predictors of a pneumococcal etiology in community-acquired pneumonia (CAP) in hospitalized children. METHODS: Children requiring hospitalization for CAP were prospectively enrolled. The following indices were determined: antibodies against pneumococcal surface proteins (anti-PLY, pneumococcal histidine triad D, pneumococcal histidine triad E, LytB and pneumococcal choline-binding protein A), viral serology, nasopharyngeal cultures and polymerase chain reaction for 13 respiratory viruses, blood pneumococcal polymerase chain reaction, pneumococcal urinary antigen, PCT and CRP. Presumed pneumococcal CAP (P-CAP) was defined as a positive blood culture or polymerase chain reaction for Streptococcus pneumoniae or as a pneumococcal surface protein seroresponse (≥2-fold increase). RESULTS: Seventy-five patients were included from which 37 (49%) met the criteria of P-CAP. Elevated PCT and CRP values were strongly associated with P-CAP with odds ratios of 23 (95% confidence interval: 5-117) for PCT and 19 (95% confidence interval: 5-75) for CRP in multivariate analysis. The sensitivity was 94.4% for PCT (cutoff: 1.5 ng/mL) and 91.9% for CRP (cutoff: 100 mg/L). A value≤0.5 ng/mL of PCT ruled out P-CAP in >90% of cases (negative likelihood ratio: 0.08). Conversely, a PCT value≥1.5 ng/mL associated with a positive pneumococcal urinary antigen had a diagnostic probability for P-CAP of almost 80% (positive likelihood ratio: 4.59). CONCLUSIONS: PCT and CRP are reliable predictors of P-CAP. Low cutoff values of PCT allow identification of children at low risk of P-CAP. The association of elevated PCT or CRP with a positive pneumococcal urinary antigen is a strong predictor of P-CAP.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin ß (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.