William E. Brown

Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein.

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.

Clara cell 10 kDa protein (CC10): Comparison of structure and function to uterogloblin

The cellular localization, functional activities and structures of rat and human Clara cell 10 kDa proteins (CC10) are compared to rabbit uteroglobin. CC10 is present exclusively in the non-ciliated cells of the surface epithelium of the pulmonary airways, whereas uteroglobin is reported to be present in the lung and reproductive organs. There is about 55% identity between the amino acid sequences of rat CC10 and either rabbit uteroglobin or human CC10. The latter two have 61% identity. Using the known structure of uteroglobin as the model, correlations between the structure and function for this group of proteins are made. Substitution of the residues for the rat and human CC10 into the structure of uteroglobin suggests that these proteins may be members of a structurally homologous family. Some of the functional differences may be due to distortion of the hydrophobic pocket in the dimeric protein and a surface hypervariability located on one contiguous helix and beta turn. Rat CC10 and rabbit uteroglobin both, nearly equally, inhibit papain and bind progesterone. Human CC10 does not inhibit papain and has markedly lower progesterone binding (4.6% of rabbit uteroglobin). Antiinflammatory activity of synthetic peptides corresponding to a homologous sequence region of uteroglobin and the two Clara cell proteins was tested. The region chosen has sequence similarity to lipocortin I. The peptides not only failed to inhibit carrageenan-induced foot pad swelling but exacerbated it. All three proteins inhibit pancreatic phospholipase A2. The phospholipase A2 inhibitory effect of CC10 may be important in regulating the inflammatory responses in the lung.

Amino-acid and cDNA nucleotide sequences of human Clara cell 10 kDa protein

A human lung cDNA expression library was screened by using a rabbit antiserum specific for a human Clara cell 10 kDa protein. The cDNA from two positive clones was sequenced by the dideoxy chain termination method. The nucleotide and primary amino-acid sequence deduced therefrom are presented. The N-terminal amino-acid sequence of the Clara cell 10 kDa protein, purified from bronchoalveolar lavage, was also determined. The deduced and experimentally determined sequences were identical where data for both were available. From the amino-acid composition, deduced and experimentally determined amino-acid sequences, it was determined that the 10 kDa protein in bronchoalveolar lavage consists of two identical 70-amino-acid long polypeptide chains joined by two cystine residues. The size of mRNA for the protein was found to be about 0.6 kb and the monomeric nascent protein, obtained by in vitro translation of lung mRNA was about 7.3 kDa in size. The 10 kDa protein recovered from bronchoalveolar lavage has 61% sequence identity with rabbit uteroglobin, the two proteins have common predicted secondary structures with marked surface differences when comparing predicted and actual structure determined by X-ray diffraction. The differences imply similarity of structure but, not identity of function.

Mouse Clara Cell 10-kDa (CC10) Protein: cDNA Nucleotide Sequence and Molecular Basis for the Variation in Progesterone Binding of CC10 from Different Species

A protein similar to the rat Clara cell 10-kDa protein (CC10) was isolated from mouse lung homogenate by conventional chromatography. cDNA for the mouse CC10 protein was identified in the mouse lung cDNA library by using radiolabeled rat CC10 cDNA as the probe. The isolated cDNA was sequenced and the deduced primary amino acid sequence was compared to the known sequences of rabbit and hare uteroglobins and human and rat CC10 proteins. The cDNA sequence was confirmed by N-terminal amino acid sequencing of the purified protein. The purified mouse CC10 was tested for its ability to bind progesterone, and the binding was found to be 27% lower than rat CC10 and 48% lower than rabbit uteroglobin. The relative binding of mouse, rat, and human CC10 may reflect subtle structural perturbations. The only notable difference between mouse and rat CC10 is in the beta bend between helices 1 and 2, at residue 16. This difference also exists between rat and human CC10. The mouse CC10 sequence compares favorably with human CC10, which does not bind progesterone; however, the mouse CC10 does not contain M60, which has been proposed to block the binding of progesterone with human CC10. The wide variation in progesterone binding among this family of proteins casts doubt on the importance of such binding as a physiologic function.

Uptake and distribution of 14C during and following inhalation exposure to radioactive toluene diisocyanate

Inhalation of toluene diisocyanate (TDI) results in toxic responses ranging from pulmonary irritation to immunological sensitization. The use of radioactively labeled isocyanate has made it possible to follow the initial uptake of the compound into the bloodstream independent of the final fate of the isocyanate. This study shows that the rate of uptake into the blood is linear during exposure to concentrations ranging from 0.00005 to 0.146 ppm and that the uptake continues to increase slightly postexposure. It also demonstrates that the radioactivity clears from the bloodstream to a level corresponding to approximately a 100 nM concentration of tolyl group after 72 hr and persists at a nanomolar level even 2 weeks following the exposure. This is similar to the response previously reported by this group for radioactively labeled methyl isocyanate. The initial rate of 14C uptake is also a linear function of the concentration of TDI when expressed either as concentration (ppm) or as concentration multiplied by duration of exposure (ppm.hr). This is discussed in comparison with the toxic responses as a function of both ppm and ppm.hr. Finally, the inclusion of the data on methyl isocyanate indicates that the uptake into arterial blood is a function of exposure concentration, independent of isocyanate structure.

Distribution and reactivity of inhaled 14C-labeled toluene diisocyanate (TDI) in rats

Inhalation exposure to toluene diisocyanate (TDI) can result in a variety of airway diseases. Concern has been expressed that a putative carcinogenic potential of TDI exists as a result of the formation of toluenediamine (TDA) by hydrolysis of the isocyanate in the body. Results from long-term bioassays (TDI inhalation versus gavage in rats and mice) are contradictory and discrepancies do exist concerning the interpretation of adverse effects. This study was performed to analyze the distribution and reactivity of radioactively-labeled TDI using vapor exposure in a rat model system. Rats were exposed to 14C-TDI vapors at concentrations ranging from 0.026 to 0.821 ppm for 4h. All tissues examined showed detectable quantities of radioactivity, with the airways, gastrointestinal system and blood having the highest levels which increased with exposure concentration. The concentration of radioactivity in the bloodstream after exposure was linear with respect to dose. The majority (74–87%) of the label associated with the blood was recovered in the plasma, and of this, 97–100% of the 14C existed in the form of biomolecular conjugates. Analysis of stomach contents shows that the majority of the label is also associated with high (>10 kDa) molecular weight species. While a larger percentage (28%) of the label is found in the low molecular weight fraction relative to blood, this low molecular weight labeled material represents at least eight different components. Thus, over the vapor exposure concentrations and time tested, it appears that conjugation is the predominant reaction and that free TDA is not a primary in vivo reaction product under the conditions tested.

Uptake and distribution of 14C during and following exposure to [14C]methyl isocyanate.

Guinea pigs were exposed to [14C]methyl isocyanate (14CH3-NCO, 14C MIC) for periods of 1 to 6 hr at concentrations of 0.5 to 15 ppm. Arterial blood samples taken during exposure revealed immediate and rapid uptake of 14C. Clearance of 14C was then gradual over a period of 3 days. Similarly 14C was present in urine and bile immediately following exposure, and clearance paralleled that observed in blood. Guinea pigs fitted with a tracheal cannula and exposed while under anesthesia showed a reduced 14C uptake in blood indicating that most of the 14C MIC uptake in normal guinea pigs occurred from retention of this agent in the upper respiratory tract passages. In exposed guinea pigs 14C was distributed to all examined tissues. In pregnant female mice similarly exposed to 14C MIC, 14C was observed in all tissues examined following exposure including the uterus, placenta, and fetus. While the form of 14C distributed in blood and tissues has not yet been identified, these findings may help to explain the toxicity of MIC or MIC reaction products on organs other than the respiratory tract, as noted by several investigators.

Inhibition of cholinesterase activity by isocyanates

Exposure of workers to isocyanates may result in irritation and/or sensitization of the respiratory tract. An immunologic mechanism for sensitization has been presented previously. This investigation explored whether, as a possible mechanism for the irritation reaction, the toxic respiratory effect of isocyanates might be due to their ability to inhibit cholinesterases. Hexamethylene diisocyanate (HDI), hexyl isocyanate (HI), and 2,6-toluene diisocyanate (2,6-TDI) were found to completely inhibit purified human serum cholinesterase when added at molar ratios of 4:1 to 8:1 (isocyanate:enzyme). By contrast, molar ratios of 50:1 or greater were required for 50% enzyme inhibition by 2,4-toluene diisocyanate (2,4-TDI), phenyl isocyanate, or o-tolyl isocyanate. Enzyme inhibition was also achieved by exposure of purified cholinesterase to atmospheres containing 1 ppm isocyanates. Under these conditions, HDI and HI were again the most potent enzyme inhibitors with much less reactivity shown by 2,4-TDI and 2,6-TDI. Under more physiologic conditions, when whole human plasma was the source of cholinesterase, HDI and HI were still potent enzyme inhibitors. However, with the latter two isocyanates, the molar concentrations needed to effect 50% enzyme inhibition suggested affinity labeling by these reagents. The potent cholinesterase inhibition shown by HDI and HI may offer explanation for observed respiratory symptomatology noted upon exposure to these isocyanates.

Issues in Diisocyanate Antibody Testing

Diisocyanates are used to produce a wide variety of polyurethane products; they are also recognized as an important cause of occupational asthma. Their chemical reactivity presents challenges to toxicologists and clinicians alike seeking to understand the mechanisms underlying diisocyanate asthma. In this article, we review the literature on immunoassay detection of IgE and IgG binding to diisocyanate–protein conjugates and assess the utility of such testing as a diagnostic tool and exposure indicator. Data from 29 studies of occupational exposure to diisocyanates revealed considerable variability in assay methodology and heterogeneity in the prevalence of positive antibody responses across laboratories. In studies that included both confirmed diisocyanate asthma subjects and exposed nonasthmatics, positive IgE responses identified cases with low sensitivity (18–27%), but high specificity (96–98%). Detection of IgG binding to diisocyanate conjugates is an indirect, qualitative indicator of disease status and past diisocyanate exposure. The utility of these assays is limited, however, due to a lack of (1) method standardization, (2) population norms to guide interpretation of results, and (3) demonstration that the assays improve either on disease prediction or on exposure confirmation beyond that of other indicators. Sources of assay heterogeneity are discussed and suggestions are offered for improving test performance and interpretability.

Amino acid composition of proteins eluted from polyacrylamide gels: Background considerations☆

Abstract It is shown that the contribution of the gel itself to amino acid compositions of proteins eluted from polyacrylamide gels is significant (especially at low protein-to-gel ratios), linearly related to the volume of the gel slices eluted, and, when applied as a correction to amino acid composition data, results in an enhancement of the composition determination.