Structure of the extracellular domains of the human interleukin-6 receptor alpha -chain.

Dysregulated production of IL-6 and its receptor (IL-6R) are implicated in the pathogenesis of multiple myeloma, autoimmune diseases and prostate cancer. The IL-6R complex comprises two molecules each of IL-6, IL-6R, and the signaling molecule, gp130. Here, we report the x-ray structure (2.4 A) of the IL-6R ectodomains. The N-terminal strand of the Ig-like domain (D(1)) is disulfide-bonded to domain D(2), and domains D(2) and D(3), the cytokine-binding domain, are structurally similar to known cytokine-binding domains. The head-to-tail packing of two closely associated IL-6R molecules observed in the crystal may be representative of the configuration of the physiological dimer of IL-6R and provides new insight into the architecture of the IL-6R complex.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Identification of ligand-binding site III on the immunoglobulin-like domain of the granulocyte colony-stimulating factor receptor.

The granulocyte colony-stimulating factor receptor (G-CSF-R) forms a tetrameric complex with G-CSF containing two ligand and two receptor molecules. The N-terminal Ig-like domain of the G-CSF-R is required for receptor dimerization, but it is not known whether it binds G-CSF or interacts elsewhere in the complex. Alanine scanning mutagenesis was used to show that residues in the Ig-like domain of the G-CSF-R (Phe(75), Gln(87), and Gln(91)) interact with G-CSF. This binding site for G-CSF overlapped with the binding site of a neutralizing anti-G-CSF-R antibody. A model of the Ig-like domain showed that the binding site is very similar to the viral interleukin-6 binding site (site III) on the Ig-like domain of gp130, a related receptor. To further characterize the G-CSF-R complex, exposed and inaccessible regions of monomeric and dimeric ligand-receptor complexes were mapped with monoclonal antibodies. The results showed that the E helix of G-CSF was inaccessible in the dimeric but exposed in the monomeric complex, suggesting that this region binds to the Ig-like domain of the G-CSF-R. In addition, the N terminus of G-CSF was exposed to antibody binding in both complexes. These data establish that the dimerization interface of the complete receptor complex is different from that in the x-ray structure of a partial complex. A model of the tetrameric G-CSF.G-CSF-R complex was prepared, based on the viral interleukin-6.gp130 complex, which explains these and previously published data.

Identification and characterization of Harc, a novel Hsp90-associating relative of Cdc37.

Although little is known about the precise mechanisms by which the molecular chaperone Hsp90 recognizes its client proteins, Cdc37 has been shown to play a critical role in the targeting of Hsp90 to client protein kinases. Described here is the identification and characterization of a novel 35-kDa human protein that is 31% identical to Cdc37. We have named this novel protein Harc (Hsp90-associating relative of Cdc37). Northern blot analysis revealed the presence of Harc mRNA in several human tissues, including liver, skeletal muscle, and kidney. Biochemical fractionation and immunofluorescent localization of epitope-tagged Harc (i.e. FLAG-Harc) indicated that it is present in the cytoplasm of cells. FLAG-Harc binds Hsp90 but unlike Cdc37 does not bind Src family kinases or Raf-1. Mapping experiments indicate that the central 120 amino acids of both Harc and Cdc37 constitute a Hsp90-binding domain not described previously. FLAG-Harc is basally serine-phosphorylated and hyperphosphorylated when co-expressed with an activated mutant of the Src family kinase Hck. Notably, FLAG-Harc forms complexes with Hsp90, Hsp70, p60Hop, immunophilins, and an unidentified p22 protein but not with the Hsp90 co-chaperone p23. Thus Harc likely represents a novel participant in Hsp90-mediated protein folding, potentially targeting Hsp90 to Hsp70-client protein heterocomplexes.

The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man.

Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where the VEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man.

Determination of the disulfide structure and N-glycosylation sites of the extracellular domain of the human signal transducer gp130.

gp130 is the common signal transducing receptor subunit for the interleukin-6-type family of cytokines. Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys(279) and Cys(469)), and one (Cys(397)) is modified by S-cysteinylation. Of the 11 potential N-glycosylation sites, Asn(21), Asn(61), Asn(109), Asn(135), Asn(205), Asn(357), Asn(361), Asn(531), and Asn(542) are glycosylated but not Asn(224) and Asn(368). The disulfide bonds, Cys(112)-Cys(122) and Cys(150)-Cys(160), are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys(6)-Cys(32) and Cys(26)-Cys(81)) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys(436)-Cys(444). We have created a model predicting that this loop maintains Cys(469) in a reduced form, available for ligand-induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130.

Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells.

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.

Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study.

Micronuclei (MN) are routinely enumerated in mouse peripheral blood to index genotoxicity. Recent data from the Collaborative Study Group for the Micronucleus Test (CSGMT) [CSGMT (The Collaborative Study Group for the Micronucleus Test), Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS MMS, Environ. Mol. Mutagen. 32 (1998) 84-100] suggest that rat peripheral blood may also be appropriate for the enumeration of MN, if scoring is limited to the youngest fraction of reticulocytes. The experiments described herein were designed to test whether modifications to a flow cytometric scoring procedure for measuring micronucleated reticulocytes (MN-RET) in mouse peripheral blood could be extended to accurately enumerate MN in rat peripheral blood. Rats were treated with saline or one of three genotoxic agents (6-mercaptopurine, ethyl methanesulfonate or propane sultone) in an acute dosing protocol. Peripheral blood samples were subsequently collected for both microscopic and flow cytometric analysis. Micronucleus frequencies were scored in the youngest fraction of reticulocytes: scoring by microscopy was restricted to the types I and II reticulocytes based on RNA content utilizing acridine orange supravital staining; flow cytometric measurements were restricted to the youngest fraction of reticulocytes based on transferrin receptor (CD71) staining. A statistically significant dose-related increase in the incidence of MN was observed, irrespective of scoring method. A higher level of statistical discrimination between control and genotoxin-treated groups was observed for the flow cytometric data and can most likely be explained by the increased number of cells scored (10x more than microscopy) and the lower scoring variability. Together, these data suggest that (i) rat peripheral blood represents an appropriate compartment for evaluating genotoxin-induced MN when the analysis is restricted to young reticulocytes, and (ii) the measurement of MN in rat peripheral blood reticulocytes benefits from the high throughput methodology of flow cytometry.

Malaria-infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry.

A procedure for optimizing the configuration of flow cytometers for enumerating micronucleated erythrocytes is described. The method is based on the use of a biological model for micronucleated erythrocytes, the malaria parasite Plasmodium berghei. P. berghei endows target cells of interest (erythrocytes) with a micronucleus-like DNA content. Unlike micronuclei, parasitized red blood cells have a homogenous DNA content, and can be very prevalent in circulation. These characteristics make malaria-infected erythrocytes extremely well suited for optimizing instrument setup on a daily basis. The experiment described herein was designed to test the hypothesis that malaria-infected erythrocytes can greatly enhance the consistency with which flow cytometers are configured for micronucleus analyses, and thereby minimize intra- and interexperimental variation. Data collected over the course of several months, on two different flow cytometers, supports the premise that malaria-infected blood represents a useful biological standard which helps ensure reliable and consistent flow cytometric enumeration of rare micronucleated erythrocytes.

Disulfide bond structure and N-glycosylation sites of the extracellular domain of the human interleukin-6 receptor.

The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular "soluble" part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77 with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potential N-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226.

An automated method for discriminating aneugen- vs. clastogen-induced micronuclei.

A flow cytometric (FCM) procedure for quantitating micronucleated reticulocytes in mouse peripheral blood samples was evaluated for its ability to discriminate between aneugen- and clastogen-induced micronuclei (MN). In this experiment, BALB/c mice were injected with 0.9% saline, the model clastogen methyl methanesulfonate (100 mg/kg bw) or the aneugen vincristine (0.2 mg/kg bw). Peripheral blood samples were collected 48 hr after injection and were subsequently fixed and stained for flow cytometric analysis. The staining method utilized FITC-conjugated anti-CD71 to differentially label reticulocytes, and the nucleic acid dye propidium iodide to resolve erythrocyte populations with and without micronuclei. The frequency of micronucleated reticulocytes was determined by analyzing 10,000 total reticulocytes per blood sample. A second analysis was performed on each sample whereby the propidium iodide associated fluorescent signals of 250 MN were collected and graphed as a single-parameter histogram. The histogram statistic "median channel" was recorded for each sample and provided a quantitative description of MN distribution according to DNA content. Cumulatively, the results of this study suggest that 1) flow cytometry can be employed to measure the incidence of MN resulting from clastogenic or aneugenic activity, and 2) MN resulting from aneugens can be discriminated from those arising spontaneously or from clastogen treatment based on flow cytometric analysis of DNA content.

Developmental language disorders.

Developmental language disorders are among the most common disorders of childhood referred to the pediatric neurologist. This article presents an overview of developmental language disorders, a discussion of the definition of developmental language disorders, potential causal factors, and a description of possible subtypes of language disorders in children. The article concludes with a review of the pediatric neurologist's role in developmental language disorders and recommendations for assessment and management.

Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase.

We describe here a sub-family of enzymes related both structurally and functionally to N-acetylneuraminate lyase. Two members of this family (N-acetylneuraminate lyase and dihydrodipicolinate synthase) have known three-dimensional structures and we now proceed to show their structural and functional relationship to two further proteins, trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and D-4-deoxy-5-oxoglucarate dehydratase. These enzymes are all thought to involve intermediate Schiff-base formation with their respective substrates. In order to understand the nature of this intermediate, we have determined the three-dimensional structure of N-acetylneuraminate lyase in complex with hydroxypyruvate (a product analogue) and in complex with one of its products (pyruvate). From these structures we deduce the presence of a closely similar Schiff-base forming motif in all members of the N-acetylneuraminate lyase sub-family. A fifth protein, MosA, is also confirmed to be a member of the sub-family although the involvement of an intermediate Schiff-base in its proposed reaction is unclear.

Relationship between language and fluency in children with developmental language disorders.

The present investigation addresses two primary hypotheses: (a) that a subset of children with developmental language disorders exhibits significantly more disfluencies than other children with language disorders and (b) that differences between the disfluent and nondisfluent groups observed in fluency may be related to differences in language deficits. Spontaneous language samples from 60 preschool children with developmental language disorders were analyzed for frequency and type of disfluencies. Comparisons of the frequency of disfluencies across subjects revealed that a subset of 10 subjects exhibited significantly more disfluencies than the other subjects with language disorders. Demographic, intelligence, and language variables were compared across the two groups to determine whether such factors could account for the differences in fluency. The subjects with greater percentages of disfluencies were found to be significantly older and demonstrated significantly higher scores on two standard measures of vocabulary. These findings were interpreted in light of two models of disfluencies: the neuropsycholinguistic (Perkins, Kent, & Curlee, 1991) and Demands and Capacities (Adams, 1990; Starkweather, 1987). This suggests that some children with language disorders are at risk for fluency breakdown because of dysynchronies in the development of lexical and syntactic aspects of language or as a result of mismathces between speaking demands and capacities.

Clinical and research congruence in identifying children with specific language impairment.

This paper reports on the results of a large multicenter project designed to develop an empirically based classification of preschool children with language impairments. A clinically selected population of 252 children with specific language impairments (SLI) was used to evaluate the reliability, coverage, and usefulness of both standard clinical and research definitions of such children. Varying degrees of congruence were found between the clinically identified children with SLI and those identified as SLI using discrepancy, deficit, and standardized operational criteria. Such mismatch between the original clinical identification and more standardized operational criteria may be related to different clinical perspectives, professional training, and limited assessment measures. These results suggest that there is a significant gulf between the clinical diagnosis of children with specific language impairment and more standardized operational criteria. It is suggested that the global concept of a "specific language impairment" may not be a useful concept for either clinical or research activities.

The validity of discrepancy criteria for identifying children with developmental language disorders.

Empirical data from two studies address the clinical validity of discrepancy criteria for identification of children with developmental language disorders (DLD). Study 1 involved 256 preschoolers clinically defined as DLD and meeting inclusionary criteria for normal hearing, intellectual, neurological, and psychiatric status. Application of alternative psychometrically derived discrepancy criteria identified only 40% to 60% of the clinically defined group as language disordered. Study 2 applied nonverbal IQ-language performance discrepancy criteria to 368 eight-year-old, randomly selected control subjects, resulting in over 45% of the controls being identified as DLD. Factors contributing to underidentification in Study 1 and over-identification in Study 2 are discussed, raising questions regarding the validity of discrepancy criteria for identification of DLD children.

Completeness of case ascertainment of the State Cancer Registry.

The authors evaluated the ability of the New Jersey State Cancer Registry to detect known cancer cases during an investigation of a cancer cluster occurring in professional football players. The Registry was found to be a valuable tool for the detection of cancer cases for New Jersey residents.

Cancer by industry: analysis of a population-based cancer registry with an emphasis on blue-collar workers.

This paper uses information on occupation and industry routinely collected in a state-based cancer registry to assess potential associations between work place exposures and cancer incidence. Industry-specific proportional cancer incidence ratios (PCIR) were calculated by race and sex for all individuals and for white males with blue-collar occupations. Expected numbers of cancers were derived both from cancers occurring among all occupations and just among blue-collar occupations. This latter analysis was done as a control for differences in the prevalence of life-style habits between blue- and white-collar workers. Increased lung cancer PCIR were seen in most industries previously reported to be associated with lung cancer risk. The effects of socioeconomic status on these results are discussed. Other results include an increased ratio of melanoma in blue-collar white male rubber and plastic product workers, an increased ratio of non-Hodgkin's lymphomas in motor vehicle manufacture workers, and an increased PCIR of chronic lymphocytic leukemia in general construction workers. Uterine cancer was increased in proportion in white females for a number of industries including rubber and plastic product manufacture, apparel manufacture, and electrical equipment manufacture.

Case-control study of decaffeinated coffee consumption and pancreatic cancer.

The relationship between decaffeinated coffee consumption and pancreatic cancer was examined using data from a hospital-based case-control study of individuals aged 20-80 years in 18 hospitals in 6 United States cities, from January 1981 to December 1984. Among the males, 127 cases and 371 controls were examined, while for females, the figures were 111 and 325 for cases and controls, respectively. Decaffeinated coffee use was not associated with an increased risk of pancreatic cancer in males (odds ratio = 0.7 for 3 or more cups/day; 95% confidence interval = 0.4-1.4). For females, an elevated risk was seen for drinkers of 1-2 cups/day (odds ratio = 1.6; 95% confidence interval = 1.0-2.7), but this finding was of borderline significance and elevation in risk was not found for drinkers of 3 or more cups/day (odds ratio = 0.9; 95% confidence interval = 0.4-1.9). Cigarette smoking was significantly associated with pancreatic cancer in both males and females. Factors examined and not found to be related to pancreatic cancer included education, occupation, religion, marital status, alcohol drinking, saccharin use, height, weight 5 years before hospitalization, history of previous diseases, and residence.

A case-control study of diesel exhaust exposure and bladder cancer.

The relationship between bladder cancer and employment in occupations involving exposure to diesel exhaust was examined using data from a hospital-based case-control study of men aged 20 to 80 years in 18 hospitals in six U.S. cities, from January 1981 to May 1983. In this analysis, 194 cases and 582 controls were compared according to occupation, smoking history, alcohol and coffee consumption, and various demographic variables. No difference was found in the proportion of bladder cancer cases employed in occupations with exposure to diesel exhaust compared to controls. This relationship did not change after taking smoking habits into account. Bladder cancer cases were significantly more likely to be current smokers of cigarettes than were controls.