Relationship between genetic polymorphisms in MCP-1, CCR-2, and non-small-cell lung cancer in the Han nationality of Northern China.

Lung cancer is a common malignant tumor worldwide and is now the leading cause of cancer-related deaths. Monocyte chemoattractant protein 1 (MCP-1) and its receptor chemokine receptor 2 (CCR-2) are important chemokines. We examined the polymorphisms of 338 unrelated patients with non-small cell lung carcinoma (NSCLC) and 200 unrelated healthy controls of Han nationality in Northern China using polymerase chain reaction-restriction fragment length polymorphism. We found a significant increase in the frequency of the MCP-1 AA genotype [0.293 vs 0.195, odds ratio (OR) = 1.71, 95% confidence interval (CI) = 1.13-2.60] and a significant decrease in the frequency of the GG genotype (0.290 vs 0.41, OR = 0.64, 95%CI = 0.47-0.87) in NSCLC patients compared to controls. The frequencies of AA-ww (0.151 vs 0.090, P = 0.041, OR = 1.80, 95%CI = 1.33-2.43) and AA-wm (0.136 vs 0.080, P = 0.049, OR = 1.81, 95%CI = 1.01-3.27) were higher in lung cancer patients than in healthy controls; the frequency of GG-wm (0.121 vs 0.190, P = 0.030, OR = 0.60, 95%CI = 0.38-0.95) was lower in lung cancer patients than in healthy controls. Based on these results, the polymorphism in MCP-1 may be correlated with the development of NSCLC in the Han nationality of Northern China. However, the polymorphism in CCR-2 is not involved in NSCLC.

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits.

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.

Microtubules containing acetylated alpha-tubulin in mammalian cells in culture.

The subcellular distribution of microtubules containing acetylated alpha-tubulin in mammalian cells in culture was analyzed with 6-11B-1, a monoclonal antibody specific for acetylated alpha-tubulin. Cultures of 3T3, HeLa, and PtK2 cells were grown on coverslips and observed by immunofluorescence microscopy after double-staining by 6-11B-1 and B-5-1-2, a monoclonal antibody specific for all alpha-tubulins. The antibody 6-11B-1 binds to primary cilia, centrioles, mitotic spindles, midbodies, and to subsets of cytoplasmic microtubules in 3T3 and HeLa cells, but not in PtK2 cells. These observations confirm that the acetylation of alpha-tubulin is a modification occurring in different microtubule structures and in a variety of eukaryotic cells. Some features of the acetylation of cytoplasmic microtubules of mammalian cells are also described here. First, acetylated alpha-tubulin is present in microtubules that, under depolymerizing conditions, are more stable than the majority of cytoplasmic microtubules. In addition to the specific microtubule frameworks already mentioned, cytoplasmic microtubules resistant to nocodazole or colchicine, but not cold-resistant microtubules, contain more acetylated alpha-tubulin than the rest of cellular microtubules. Second, the alpha-tubulin in all cytoplasmic microtubules of 3T3 and HeLa cells becomes acetylated in the presence of taxol, a drug that stabilizes microtubules. Third, acetylation and deacetylation of cytoplasmic microtubules are reversible in cells released from exposure to 0 degrees C or antimitotic drugs. Fourth, the epitope recognized by the antibody 6-11B-1 is not absolutely necessary for cell growth and division. This epitope is absent in PtK2 cells. The acetylation of alpha-tubulin could regulate the presence of microtubules in specific intracellular spaces by selective stabilization.

Contractile proteins in Drosophila development.

In summary, we have used a multidisciplinary approach to the analysis of actomyosin-based motility during Drosophila embryogenesis. We have documented the movements of early embryogenesis with modern, video methods. We have characterized the cytoplasmic myosin polypeptide, made specific polyclonal antisera to the molecule, studied its distribution during early embryogenesis, cloned and partially characterized the gene that encodes it, and have recently completed the nucleotide sequence of a nearly full length cDNA that encodes the entire protein-coding region. We have initiated studies on myosin function in living embryos both by direct microinjection of antibodies and through classical genetics. To better understand how myosin function is regulated, we have begun analysis of its light chains. Finally, to investigate the molecular mechanism by which its function is integrated into a labile cytoskeleton, whose architecture is constantly changing, we have also investigated Drosophila spectrins. Together, these studies are designed to shed light on the dynamics of biologic form at the cellular level, with current focus on such complex processes as cytokinesis and morphogenesis.

Application of a macroporous resin containing imidazoline groups to preconcentration and separation of gold, platinum and palladium prior to ICP-AES determination.

A new functional resin with a long functional side chain was synthesized by modification of aminated macroporous poly(vinyl chloride) resin with cyanoethylene and ethylenediamine. Traces of Au(III), Pt(IV) and Pd(II) in aqueous solution were quantitatively adsorbed in the acidity range of pH 4 and C(H(+)) 3 M. The rate of equilibration is high; Cu(2+), Fe(3+), Ni(2+), etc. exhibit little interference on the adsorption of the sought noble metals. The saturated adsorption capacities for Au(III), Pt(IV), Pd(II) and Ir(IV) in 2 M HCl were 4.0, 1.57, 2.26, 1.85 mmol g(-1). Adsorbed ions can be quantitatively desorbed by 4% thiourea +0.25 M H(2)SO(4). The resin has good reusability, and can be used for preconcentration and separation of Au(III), Pt(IV) and Pd(II) prior to their determination by ICP-AES with satisfactory results.

Essential light chain of Drosophila nonmuscle myosin II.

We have cloned and sequenced a cDNA encoding the essential (alkaline) light chain of nonmuscle myosin from Drosophila melanogaster. The protein predicted from the cDNA matches partial amino acid sequence derived from essential light chain protein that copurifies with native nonmuscle myosin heavy chain. This completes the sequence of the three myosin subunits, two of which have been shown genetically to be required for morphogenesis and cytokinesis (the heavy chain encoded by zipper and the regulatory light chain encoded by spaghetti squash). The essential light chain protein is 147 amino acids in length and is 53% identical to human smooth muscle essential light chain. The sequence is consistent with the presence of four helix-loop-helix domains seen in crystallographic structures of the striated muscle myosin light chains and their close relative, calmodulin. We identified the most conserved residues among essential light chain sequences from multiple phyla and present their locations on the crystallographic structure of striated muscle essential light chain. This highlights several conserved contacts among the myosin subunits that may be important for the structure and regulation of the myosin motor. The gene encoding Drosophila nonmuscle essential light chain (Mlc-c) localizes to cytological position 5A6 and we discuss prospects for genetic analysis in this region.

The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila.

Two independent approaches to understanding the molecular mechanism of cytokinesis have converged on the gene spaghetti-squash (sqh). A genetic screen for mitotic mutants identified sqh1, a mutation that disrupts cytokinesis, which was then cloned by transposon tagging. Independently, the gene that encodes the regulatory light chain of the biochemically defined nonmuscle myosin (MRLC-C) was also cloned. We show here that sqh encodes MRLC-C and that in sqh1 mutants, the level of stable light chain transcript is greatly reduced. Reversion by transposon excision or transformation with a wild-type copy of the sqh transcription unit rescues cytokinesis failure and other defects in sqh1. Vertebrate homologs of MRLC-C are phosphorylatable and regulate myosin activity in vitro. These studies provide genetic proof that MRLC-C is required for cytokinesis, suggest a role for the protein in regulating contractile ring function, and establish a genetic system to evaluate its function.

Molecular characterization of cytosolic phospholipase A2-beta.

We have isolated a cDNA encoding a 1012-amino acid polypeptide cPLA2-beta, that has significant homology with cPLA2-alpha in both the calcium-dependent lipid binding domain as well as in the catalytic domain. Transient expression of cPLA2-beta cDNA in COS cells results in an increase in calcium-dependent phospholipase A1 (PLA1) and PLA2 activities compared with vector-transfected cells. cPLA2-beta is markedly less selective for cleavage at sn-2 as compared with cPLA2-alpha and cPLA2-gamma. Northern analysis reveals a cPLA2-beta transcript of 8 kilobase pairs that is expressed in all the human tissues examined. With the identification of cPLA2-beta, the newly defined cPLA2 family now comprises three members that may have dramatically different mechanisms for regulation of expression and enzymatic activation.

Serum and erythrocyte amino acid pattern: studies on major burn cases.

Venous serum amino acids were measured in 13 patients with major burns. Erythrocyte amino acids and plasma cortisol, blood sugar and urine catecholamine were measured in two representative subgroups respectively. After burn injury, serum proline, glycine, valine, isoleucine and arginine were significantly decreased; phenylalanine, cysteine, methionine, leucine, glutamate, alanine, aspartic acid and tyrosine were significantly increased. Histidine and lysine fluctuated. This serum amino acid profile is considered as a specific pattern for major burns. Serum phenylalanine was markedly elevated in the hypermetabolic burn patients, its fluctuation coincided with the burn course and was negatively correlated with serum albumin level (P less than 0.001). These findings suggest that the ratio of phenylalanine tyrosine is a useful clinical parameter for assessing the patient's nutritional condition. Twenty-three simultaneous determinations of both serum and erythrocyte amino acid concentrations show similar changes, suggesting that the serum amino acid profile might reflect the change of total free amino acid pool. After burn injury, plasma cortisol, blood sugar and urine catecholamine were elevated as well as urine urea nitrogen. However, although the first three returned to normal by the end of the second week post burn, urine urea nitrogen remained high. This indicates that there are other factors controlling nitrogen loss in patients with major burns, it is also postulated that, due to the abnormal amino acid pattern revealed after major burns, the constituents of commercially available amino acid solutions should be modified.

Changes of serum amino acids in severely burned patients.

The serum amino acids profile in ten severe burn patients was basically similar with the findings in major burns reported in our proceeding article, supporting the conclusion that burn patients might have a particular amino acid pattern. The larger was the burn size, the more severe was the nitrogen loss. Following a severe burn, the patient was faced with the challenge of acute protein malnutrition. After severe burns, the ratio of serum Phe/Tyr rose to a higher level than in the major burns. Moreover, the elevation of serum Met/Cys ratio indicated a more serious metabolic disturbance. During the first two weeks postburn, acute decrease of serum BCAA by 20-30 per cent of the normal value was associated with a striking increase of mortality. This fact indicated the level of BCAA might be of prognostic value. In severe burns, other than huge amount of calories and protein supplied, enriched BCAA, and perhaps, carnitine might be beneficial.

A novel calcium-independent phospholipase A2, cPLA2-gamma, that is prenylated and contains homology to cPLA2.

We report the cloning and characterization of a novel membrane-bound, calcium-independent PLA2, named cPLA2-gamma. The sequence encodes a 541-amino acid protein containing a domain with significant homology to the catalytic domain of the 85-kDa cPLA2 (cPLA2-alpha). cPLA2-gamma does not contain the regulatory calcium-dependent lipid binding (CaLB) domain found in cPLA2-alpha. However, cPLA2-gamma does contain two consensus motifs for lipid modification, a prenylation motif (-CCLA) at the C terminus and a myristoylation site at the N terminus. We present evidence that the isoprenoid precursor [3H]mevalonolactone is incorporated into the prenylation motif of cPLA2-gamma. Interestingly, cPLA2-gamma demonstrates a preference for arachidonic acid at the sn-2 position of phosphatidylcholine as compared with palmitic acid. cPLA2-gamma encodes a 3-kilobase message, which is highly expressed in heart and skeletal muscle, suggesting a specific role in these tissues. Identification of cPLA2-gamma reveals a newly defined family of phospholipases A2 with homology to cPLA2-alpha.

P-selectin glycoprotein ligand-1 is the major counter-receptor for P-selectin on stimulated T cells and is widely distributed in non-functional form on many lymphocytic cells.

P-selectin glycoprotein ligand-1 (PSGL-1) is the high affinity counter-receptor for P-selectin on myeloid cells (Sako, D., Chang, X.J., Barone, K.M., Vachino, G., White, H.M., Shaw, G., Veldman, G.M., Bean, K.M., Ahern, T.J., Furie, B., Cumming, D. A., and Larsen, G. R. (1993) Cell 75, 1179-1186). Here we demonstrate that PSGL-1 is also widely distributed on T- and B-lymphocytic tumor cell lines, resting peripheral blood T and B cells, and on stimulated peripheral blood T cell and intestinal intraepithelial lymphocyte (IEL) lines. However, the majority of PSGL-1-positive resting peripheral blood lymphocytic cells and lymphoid tumor cell lines do not display significant P-selectin binding. In contrast, in vitro stimulated peripheral blood T cell and IEL lines avidly bind P-selectin, and PSGL-1 is the sole high affinity counter-receptor mediating this binding. During the course of in vitro stimulation, cell surface expression levels of PSGL-1 do not change as P-selectin binding increases. Rather, the activities of two glycosyltransferases reportedly involved in the production of functional PSGL-1 in myeloid cells are substantially higher in the stimulated T-lymphocytic lines than in resting T lymphocytes, consistent with the hypothesis that activation-dependent post-translational events contribute to the expression of functional PSGL-1 on lymphocytes.

Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro.

We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E. coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.

Leukocyte recruitment into human skin transplanted onto severe combined immunodeficient mice induced by TNF-alpha is dependent on E-selectin.

In order to study the in vivo role of E-selectin in human inflammation, we have developed a model in which human skin is transplanted onto severe combined immunodeficient (SCID) mice. The grafted skin closely resembles normal skin and retains its human vasculature. After intradermal injection of rTNF-alpha, human E-selectin was rapidly up-regulated on dermal microvessels, with significant expression (determined immunohistochemically) at 1 h postinjection and maximum expression at 2 h postinjection. To study the functional role of E-selectin, a murine Ab against human E-selectin (mAb HEL 3/2) was developed that inhibited the in vitro adhesion of both human U937 cells and murine 32D cells to TNF-alpha-stimulated human endothelial cells. After intradermal injection of TNF-alpha, large numbers of murine leukocytes migrated into the grafts within 2 h. Intravenous injection of the antihuman E-selectin mAb 3/2 completely inhibited murine white blood cell (WBC) transmigration into the skin grafts, but an isotype-matched control Ab that also bound to human endothelium had no effect. Antihuman E-selectin mAb 3/2 was also able to inhibit the migration of i.v. 51Cr-labeled human neutrophils. These findings demonstrate that E-selectin is important in early white blood cell adhesion events and is required for TNF-alpha-induced white blood cell transmigration in the human/SCID mouse chimeric model.

Expression cloning of a functional glycoprotein ligand for P-selectin.

The initial adhesive interactions between circulating leukocytes and endothelia are mediated, in part, by P-selectin. We now report the expression cloning of a functional ligand for P-selectin from an HL-60 cDNA library. The predicted amino acid sequence reveals a novel mucin-like transmembrane protein. Significant binding of transfected COS cells to P-selectin requires coexpression of both the protein ligand and a fucosyltransferase. This binding is calcium dependent and can be inhibited by a neutralizing monoclonal antibody to P-selectin. Cotransfected COS cells express the ligand as a homodimer of 220 kd. A soluble ligand construct, when coexpressed with fucosyltransferase in COS cells, also mediates P-selectin binding and is immunocrossreactive with the major HL-60 glycoprotein that specifically binds P-selectin.

Waterborne outbreak of rotavirus diarrhoea in adults in China caused by a novel rotavirus.

During two epidemics of acute diarrhoea in China in late 1982/early 1983, more than 12 000 adults in two coal mining districts were affected. The virus isolated from stool samples resembled a rotavirus morphologically. Antigenically it lacked the group antigen shared by known rotaviruses. Like other rotaviruses it had a double-stranded RNA with 11 discrete segments, but the pattern of migration of the segments on polyacrylamide electrophoresis differed from those of other rotaviruses.