Biomedical subjects

J Sun

Publications and source records attributed to J Sun.

At least 433 records · Page 24Linked to original sources

PubMed1997

Expression, reconstitution and characterization of prolixin-S as a vasodilator--a salivary gland nitric-oxide-binding hemoprotein of Rhodnius prolixus.

Prolixin-S, an anticoagulant from the salivary gland of the blood-sucking insect Rhodnius prolixus is also one of the members of salivary gland hemoproteins. We produced recombinant protein using a baculovirus-insect cell expression system, reconstituted the hemoprotein and made some characterization of it as a nitric oxide carrier. The reconstituted protein exhibited the absorption spectrum of a high-spin ferric hemoprotein with a Soret absorption peak at 400 nm. By binding nitric oxide (NO-prolixin-S), the Soret band shifted from 400 nm to 420 nm and two sharp bands (Q bands, at 535 nm and 565 nm) also appeared in the visible region. In a bioassay with aortic smooth muscle, NO-prolixin-S showed strong relaxation activity in a dose-dependent manner, which demonstrated that prolixin-S really acts as an NO carrier. A Soret absorption change also indicated that nitric oxide was gradually released under these conditions (pH 7.4 and 37 degrees C). However, at low temperature (20 degrees C) and/or low pH (pH 6), which mimic those in the insect's salivary glands, the releasing became very slow. These different NO-binding properties would enable prolixin-S to reserve nitric oxide in the salivary glands and release it in the host's blood vessels.

Animals↗

PubMed1997

Proximity of periplasmic loops in the lactose permease of Escherichia coli determined by site-directed cross-linking.

Out of over 60 single-Cys mutants in putative periplasmic loops in lactose permease, three mutants [Tyr101 --> Cys (loop III/IV), Leu313 --> Cys (loop IX/X), and Ser375 --> Cys (loop XI/XII)] spontaneously form disulfide-linked dimers, indicating that these loops are located on the periphery of the 12-helix bundle that comprises the permease. By using a permease construct with a factor Xa protease site in the middle cytoplasmic loop, cross-linking between paired-Cys residues in the N- and C-terminal halves of the permease was studied by spontaneous or copper-(1, 10-phenanthroline)3-catalyzed disulfide formation or by cross-linking with homo- or heterobifunctional reagents in which the distance between the reactive groups and the flexibility of the linker vary. The findings suggest that the longer loops are relatively flexible; however, cross-linking of residues between loops is specific, indicating that these domains are not simply flexible, hydrophilic connections between helices that interact randomly. More specifically, the findings indicate that the first periplasmic loop (loop I/II) is close to loops VII/VIII and XI/XII, placing helix XII in close proximity to helices II and XI. In addition, the observations are consistent with previous results [Wu, J., & Kaback, H. R. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 14498-502] demonstrating that helices I and II are close to helices VII and XI. Finally, evidence is presented indicating that conformational flexibility between loops I/II and XI/XII may be important for permease turnover.

Binding Sites↗

PubMed1997

Recombinant caspase-3 expressed in Pichia pastoris is fully activated and kinetically indistinguishable from the native enzyme.

Intracellular cysteine proteinases (caspases) play key roles in inflammation and apoptosis. Recombinant caspases are typically produced in Escherichia coli expression systems with the attendant problems of solubilization, re-folding and activation of the protease. Here we describe the expression of hexahistidine-tagged caspase-3 (CPP32/Yama/Apopain) in the methylotropic yeast Pichia pastoris, and the purification of soluble enzyme from yeast lysates using cobalt affinity chromatography. The recombinant protease is fully activated, stable, and cleaves the synthetic substrate DEVD-AFC (Km 16.8 microM) but not YVAD-AFC. It mediates the cleavage of the apoptotic death substrate poly(ADP-ribose) polymerase in cell extracts, but does not cleave pro-interleukin-1beta. It is inhibited by the peptide DEVD-CHO (Ki 2.2 nM), far less efficiently by YVAD-CMK (Ki 0.3 microM), and not detectably by CrmA. By these criteria, recombinant caspase-3 is indistinguishable from native caspase-3 purified from apoptotic cell extracts. Activation of recombinant caspase-3 occurs in yeast in the absence of any intrinsic caspase activity, suggesting that caspase-3 can auto-activate. However, the purified enzyme was incapable of cleaving pro-caspase-3 indicating that autoactivation of caspase-3 in vivo is not likely to occur unless very high concentrations are achieved.

Caspase 3↗

PubMed1997

Role of Janus kinase/signal transducer and activator of transcription and mitogen-activated protein kinase cascades in angiotensin II- and platelet-derived growth factor-induced vascular smooth muscle cell proliferation.

In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. In the present study, we found that electroporation of antibodies against MEK1 or ERK1 abolished vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolished proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevented proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 had no effect on angiotensin II- or platelet-derived growth factor-induced Ras/Raf-1 complex formation. Our results indicate that: 1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, 2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and 3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors.

Angiotensin II↗

PubMed1997

Tyrosine residue in exon 14 of the cytoplasmic domain of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates ligand binding specificity.

Platelet/endothelial cell adhesion molecule (PECAM-1) is a cell adhesion molecule of the immunoglobulin superfamily that plays a role in a number of vascular processes including leukocyte transmigration through endothelium. The presence of a specific 19- amino acid exon within the cytoplasmic domain of PECAM-1 regulates the binding specificity of the molecule; specifically, isoforms containing exon 14 mediate heterophilic cell-cell aggregation while those variants missing exon 14 mediate homophilic cell-cell aggregation. To more precisely identify the region of exon 14 responsible for ligand specificity, a series of deletion mutants were created in which smaller regions of exon 14 were removed. After transfection into L cells, they were tested for their ability to mediate aggregation. For heterophilic aggregation to occur, a conserved 5-amino acid region (VYSEI in the murine sequence or VYSEV in the human sequence) in the mid-portion of the exon was required. A final construct, in which this tyrosine was mutated into a phenylalanine, aggregated in a homophilic manner when transfected into L cells. Inhibition of phosphatase activity by exposure of cells expressing wild type or mutant forms of PECAM-1 to sodium orthovanadate resulted in high levels of cytoplasmic tyrosine phosphorylation and led to a switch from heterophilic to homophilic aggregation. Our data thus indicate either loss of this tyrosine from exon 14 or its phosphorylation results in a change in ligand specificity from heterophilic to homophilic binding. Vascular cells could thus determine whether PECAM-1 functions as a heterophilic or homophilic adhesion molecule by processes such as alternative splicing or by regulation of the balance between tyrosine phosphorylation or dephosphorylation. Defining the conditions under which these changes occur will be important in understanding the biology of PECAM-1 in transmigration, angiogenesis, development, and other processes in which this molecule plays a role.

Alternative Splicing↗

PubMed1997

Topography of the photosystem I core proteins of the cyanobacterium Synechocystis sp. PCC 6803.

PsaA and PsaB are homologous integral membrane proteins that form the heterodimeric core of photosystem I. Domain-specific antibodies were generated to examine the topography of PsaA and PsaB. The purified photosystem I complexes from the wild type strain of Synechocystis sp. PCC 6803 were treated with eight proteases to study the accessibility of cleavage sites in PsaA and PsaB. Proteolytic fragments were identified using the information from N-terminal amino acid sequencing, reactivity to antibodies, apparent mass, and specificity of proteases. The extramembrane loops of PsaA and PsaB differed in their accessibility to proteases, which indicated the folded structure of the loops or their shielding by the small subunits of photosystem I. NaI-treated and mutant photosystem I complexes were used to identify the extramembrane loops that were exposed in the absence of specific small subunits. The absence of PsaD exposed additional proteolytic sites in PsaB, whereas the absence of PsaE exposed sites in PsaA. These studies distinguish PsaA and PsaB in the structural model for photosystem I that has been proposed on the basis of x-ray diffraction studies (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H. T., and Saenger, W. (1996) Nat. Struct. Biol. 3, 965-973). Using osmotically shocked cells for protease treatments, the N terminus of PsaA was determined to be on the n side of the photosynthetic membranes. Based on these data and available published information, we propose a topological model for PsaA and PsaB.

Animals↗

PubMed1997

A clerodane diterpene with antibacterial activity from Ajuga lupulina.

The structure of a new diterpene, C30H46O11, with antibacterial activity against Pseudomonas aeruginose and Escherichia coli, isolated from the fresh whole plants of Ajuga lupulina (Labiatae) was established to be 2 beta-hydroxy-2-methylbutanoyl-3 alpha-lupulin (3-deoxy-14,15-dihydro-2-hydroxy-15-methoxycaryoptinol 2-methylbutanoate), by means of X-ray crystallographic analysis. The present study confirms that the two six-membered rings are in ideal chair conformations.

Anti-Bacterial Agents↗

PubMed1997

A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9).

Serine proteinase inhibitors (serpins) are classically regulators of extracellular proteolysis, however, recent evidence suggests that some function intracellularly. Such "ovalbumin" serpins include the human proteinase inhibitors 6 (PI-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and the monocyte/neutrophil elastase inhibitor. PI-9 is a potent granzyme B (graB) inhibitor that has an unusual P1 Glu and is present primarily in lymphocytes. In a search for the murine equivalent of PI-9 we screened cDNA libraries, and performed reverse transcriptase-polymerase chain reaction on RNA isolated from leukocyte cell lines and from lymph nodes and spleens of allo-immunized mice. We identified 10 new ovalbumin serpin sequences: two resemble PI-8, two resemble PI-9, and the remaining six have no obvious human counterparts. By RNA analysis only one of the two sequences resembling PI-9 (designated SPI6) is present in mouse lymphocytes while the other (a partial clone designated mBM2A) is predominantly in testis. SPI6 comprises a 1.8-kilobase cDNA encoding a 374-amino acid polypeptide that is 68% identical to PI-9. mBM2A is 65% identical to PI-9 and over 80% identical to SPI6. Although the reactive loops of SPI6 and mBM2A differ from PI-9, both contain a Glu in a region likely to contain the P1-P1' bond. SPI6 produced in vitro using a coupled transcription/translation system formed an SDS-stable complex with human graB and did not interact with trypsin, chymotrypsin, leukocyte elastase, pancreatic elastase, thrombin, or cathepsin G. Recombinant SPI6 produced in a yeast expression system was used to examine the interaction with human graB in more detail. The second-order rate constant for the interaction was estimated as 8 x 10(4) M-1 s-1, and inhibition depended on the Glu in the SPI6 reactive center. The SPI6 gene was mapped to the same region on mouse chromosome 13 as Spi3, which encodes the murine homolog of PI-6. We conclude that even though their reactive centers are not highly conserved, SPI6 is a functional homolog of PI-9, and that the regulation of graB in the mouse may involve a second serpin encoded by mBM2A. Our identification of multiple sequence homologs of PI-8 and PI-9, and six new ovalbumin serpins, is consonant with the idea that the larger set of granule and other proteinases known to exist in the mouse (compared with human) is balanced by a larger array of serpins.

Amino Acid Sequence↗

PubMed1997

Resonance Raman spectral properties and stability of manganese protoporphyrin IX cytochrome b5.

The structure and stability of cytochrome b5 reconstituted with manganese protoporphyrin IX instead of iron protoporphyrin IX has been investigated by resonance Raman spectroscopy and stopped-flow visible spectroscopy. The resonance Raman spectrum of MnIII cytochrome b5 was consistent with a high-spin hexacoordinate MnIII protoporphyrin IX structure that converted to a high-spin pentacoordinate structure at higher laser power. The resonance Raman spectrum of MnII cytochrome b5 indicated a high-spin pentacoordinate structure which was independent of laser power. Studies of the binding of MnIII protoporphyrin IX to apocytochrome b5 indicated that the MnIII-containing porphyrin bound much less tightly to the protein than did heme. Although the second-order rate constant at 20 degrees C for the association of heme with apocytochrome b5 (4.5 x 10(7) M(-1) s(-1)) was estimated to be only 1 order of magnitude higher than that with Mn protoporphyrin IX (3.3 x 10(6) M(-1) s(-1)), the dissociation of manganese substituted cytochrome b5 into the apoprotein and free Mn protoporphyrin IX occurs with a first-order rate constant of 1.2 x 10(-2) s(-1) at 20 degrees C while the dissociation of heme from cytochrome b5 at room temperature occurs 3 orders of magnitude more slowly with a first-order rate constant of 1.67 x 10(-5) s(-1) [Vergeres, G., Chen, D. Y., Wu, F.F., & Waskell, L. (1993) Arch. Biochem. Biophys. 305, 231-241]. The equilibrium dissociation constant for manganese-substituted cytochrome b5 increased with temperature from 4 nM at 20 degrees C to 14 nM at 37 degrees C. These results suggest that, in the reconstituted cytochrome P450 metabolizing system, especially in studies done with low protein concentrations (0.1 microM), and at elevated temperatures (37 degrees C), as much as 30% of the manganese-substituted cytochrome b5 may dissociate to free Mn-protoporphyrin IX and apocytochrome b5.

Animals↗

PubMed1997

Role of CD4+ T cells in pathogenesis associated with Leishmania amazonensis infection.

Most inbred strains of mice are susceptible to Leishmania amazonensis infection. We have examined the mechanism(s) underlying this generalized susceptibility using mice deficient in T cell development or in the expression of either MHC class I or class II. In contrast to wild-type C57BL/6 (B6) mice that uniformly developed large ulcerating lesions, mice lacking functional CD4+ T cells (due to targeted disruption of genes for either MHC class II trans-activator or I-A beta) showed no signs of lesion development for up to 12 to 14 wk postinfection and contained significantly lower numbers of parasites in lesions. Similarly, both B6 nude and RAG2 -/- mice failed to develop lesions. However, RAG2 -/- mice reconstituted with naive wild-type CD4+ T cells and beta2m -/- mice did develop lesions. Lesions of MHC class II -/- mice contained minimal numbers of CD8+ T cells, a marked reduction of monocytes/macrophages, and evident extracellular parasites. The inability to mount an inflammatory response in MHC class II -/- mice correlated with the failure to produce lymphokines that lead to the recruitment of monocytes/granulocytes. These results demonstrate that CD4+ T cells are the primary lymphocyte subset that mediates cellular infiltration, lesion pathology, and therefore, susceptibility to L. amazonensis infection. The disease-promoting CD4+ T cells in L. amazonensis-infected mice have the characteristics of Th1 cells. The striking differences in the course of infection between MHC class II -/- mice infected with L. amazonensis and Leishmania major suggest that these parasites may have adapted different strategies regarding the CD4-dependent immune response.

Animals↗

PubMed1997

GGTI-298 induces G0-G1 block and apoptosis whereas FTI-277 causes G2-M enrichment in A549 cells.

The mechanism by which the geranylgeranyltransferase I inhibitor GGTI-298 and the farnesyltransferase inhibitor FTI-277 inhibit human tumor growth is not known. Herein, we demonstrate that in the human lung adenocarcinoma A549 cells, GGTI-298 induced a G1-G0 block whereas FTI-277 induced an enrichment in the G2-M phase of the cell cycle. Although FTI-277, GGTI-298, and compactin inhibited A549 cell growth, only GGTI-298 and compactin induced apoptosis as demonstrated by four criteria: 4',6-diamidine-2-phenylindoledihydrochloride staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, DNA fragmentation assay, and flow cytometry. Furthermore, the involvement of geranylgeranylated proteins in apoptotic pathways was confirmed by demonstrating that geranylgeraniol was able to block the ability of compactin to induce apoptosis. These results suggest that protein geranylgeranylation is critical for the control of programmed cell death and that, in A549 cells, farnesylated and geranylgeranylated proteins are involved in G2-M and G0-G1, respectively.

Adenocarcinoma↗

PubMed1997

A physico-chemical study of triple helix formation by an oligodeoxythymidylate with N3'--> P5' phosphoramidate linkages.

Non-denaturing gel retardation assay, DNA melting experiments and FTIR spectroscopy were used to characterize the triple helix formed by a 15mer 2'-deoxythymidylate with N3'-->P5'phosphoramidate linkages with its target sequence. The results indicate that: (i) the pentadecadeoxythymidylate with phosphoramidate linkages [dT15(np)] is highly potent to form a triple helix with a dT15*dA15target duplex through Hoogsteenbase-pairing; (ii) it forms a dT15(np)*dA15xdT15(np) triplex with the single-stranded oligo-2'-deoxyadenylate (dA15) without detectable double-helical intermediate; (iii) it does not only form a triple helix on the dT15*dA15target duplex, but also partially displaces the dT15 strand from the dT15*dA15duplex to form a dT15(np)*dA15xdT15(np) complex.

DNA↗

PubMed1997

Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent mitogen and chemotactic factor for fibroblasts, smooth muscle cells and keratinocytes. It is demonstrated that HB-EGF is not only a ligand for HER1, as previously reported, but for HER4 as well. HB-EGF binds to NIH 3T3 cells overexpressing either HER1 or HER4 alone, but not HER2 or HER3 alone. Binding to HER4 is independent of HER1. The ability of HB-EGF to bind to two different receptors is in contrast to EGF which binds to HER1, but not to HER4, and heregulin-beta1 which binds to HER4, but not to HER1. Besides binding, HB-EGF activates HER4. For example (i) it induces tyrosine phosphorylation of HER4 in cells overexpressing this receptor and of endogenous HER4 in MDA-MB-453 cells and astrocytes; (ii) it induces association of phosphatidylinositol 3-kinase (PI3-K) activity with HER4; and (iii) it is a potent chemotactic factor for cells overexpressing HER4. Chemotaxis is inhibited by wortmannin, a PI3-K inhibitor, suggesting a possible role for PI3-K in mediating HB-EGF-stimulated chemotaxis. On the other hand, HB-EGF is not a mitogen for cells expressing HER4, in contrast to its ability to stimulate both chemotaxis and proliferation in cells expressing HER1. It was concluded that HER4 is a newly described receptor for HB-EGF and that HB-EGF can activate two EGF receptor subtypes, HER1 and HER4, but with different biological responses.

3T3 Cells↗

PubMed1997

Regression analysis of interval-censored failure time data.

Interval-censored failure time data often occur, for example, in clinical trials or longitudinal studies. For the regression analysis of such data, there have been a number of methods proposed based on continuous regression models such as Cox's proportional hazards model. In practice, however, observed interval-censored data that arise from clinical trials are often given in a discrete scale due to the nature of clinical trials although the underlying variable may be continuous. It is apparent that in this case, one can better handle analysis of the data with the methods based on discrete models. In this paper, I propose a method based on a discrete logistic model for the regression analysis of interval-censored failure time data with focus on the comparison of failure time distributions among different treatments. I discuss the relationship between the proposed method and existing methods.

Clinical Trials as Topic↗

PubMed1997

Measurement of patient positioning errors in three-dimensional conformal radiotherapy of the prostate.

PURPOSE/OBJECTIVE: To determine the spatial distribution of setup errors for patients treated with six-field, three-dimensional (3D) conformal radiation therapy for prostate cancer. METHODS AND MATERIALS: Port films for 50 patients were analyzed retrospectively. The port films were digitized and compared, using image registration software, to simulator films (representing the ideal treatment position). Patient positioning uncertainty for a given setup was determined using port films from three projections, two obliques, and one lateral. A total of 1239 port films and 300 simulator films were analyzed for the study. Patient position was analyzed for out-of-plane rotations and time trends over the course of treatment. RESULTS: The distribution of systematic setup errors for the 50 patients, defined as the mean patient displacement for the treatment course, had a mean and standard deviation (SD) of (-0.1 +/- 1.9) mm, (0.4 +/- 1.4) mm, and (-0.3 +/- 1.3) mm in the mediolateral (ML), superior-inferior (SI) and anterior-posterior (AP) directions, and (-0.1 +/- 0.2) for rotational errors. The distribution of random setup errors about the mean approximated a normal distribution and the standard deviations for the population of patients in the ML, SI, and AP directions, were 2.0 mm, 1.7 mm, and 1.9 mm, respectively. The distribution of out-of-plane rotations had 1 SD of 0.9 degrees and 0.6 degrees about the SI and AP axes. Ten of the 50 patients demonstrated a statistically significant time trend in their setup position resulting in shifts ranging from 2 to 7 mm. CONCLUSIONS: The setup verification protocol appears to minimize systematic setup errors to a level that approaches the sensitivity of the image registration technique. The random day to day fluctuations, represented by the average values of the standard deviations, are minor in comparison to the currently used margins, which further emphasizes the effectiveness of this protocol in conjunction with the use of the immobilization device.

Analysis of Variance↗

PubMed1997

Cysteine-scanning mutagenesis of helix II and flanking hydrophilic domains in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix II and flanking hydrophilic loops (from Leu34 to Lys74) was replaced individually with Cys. Of the 41 single-Cys mutants, 28 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 10 mutants exhibit lower but significant levels of accumulation (25-60% of C-less). His35-->Cys permease exhibits very low activity (ca. 20% of C-less), while Gly64-->Cys or Asp68-->Cys permease is unable to accumulate lactose. However, His35 can be replaced with Arg without effect on transport activity [Padan, E., Sarkar, H.K., et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765-6768]. In addition, even though mutant Gly64-->Cys or Glu68-->Cys is inactive both in the C-less background and in the wild-type, neither Gly64 [Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030-1039] nor Glu68 [Jessen-Marshall, A.E., & Brooker, R.J. (1996) J. Biol. Chem. 271, 1400-1404] is essential for active lactose transport. Immunoloblot analysis reveals that all of the mutants except His35-->Cys permease are inserted into the membrane at concentrations comparable to that of C-less permease. The transport activity of the single-Cys mutants is altered by N-ethylmaleimide (NEM) treatment in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to NEM inactivation at positions that cluster in a manner indicating that they are on one face of an alpha-helix (Thr45-->Cys, Gly46-->Cys, Phe49-->Cys, Ser53-->Cys, Ser56-->Cys, Gln60-->Cys, and Ser67-->Cys). Interestingly, the same face contains positions where Cys substitution itself leads to low transport activity (Ile52-->Cys, Leu57-->Cys, Gln60-->Cys, and Gly64-->Cys). The results demonstrate that although no residue per se in this region of the permease is irreplaceable, the surface of one face of helix II is important for active lactose transport.

Amino Acid Sequence↗

PubMed1997

The last two cytoplasmic loops in the lactose permease of Escherichia coli comprise a discontinuous epitope for a monoclonal antibody.

Monoclonal antibody (mAb) 4B11 binds to a conformational epitope in the lactose permease that is exposed on the cytoplasmic face of the membrane with a KD of 2.8 x 10(-7) M. By studying binding of 4B11 to permease mutants containing six contiguous His residues in each cytoplasmic loop, inserted factor Xa protease sites, or a C-terminal deletion, the cytoplasmic loops between helices VIII and IX (loop VIII/IX) and between helices X and XI (loop X/XI) are shown to comprise the epitope. Subsequently, Cys-scanning mutagenesis in conjunction with thiol modification was carried out in order to identify specific residues involved in 4B11 recognition. Glu342 and Arg344 in loop X/XI are primary determinants for 4B11 binding, while Ile283 in loop VIII/IX and Phe334 and Lys335 in loop X/XI are secondary determinants. Consistently, binding of avidin to biotinylated single-Cys replacements in loop VIII/IX or loop X/XI blocks 4B11 binding, but avidin binding to biotinylated Cys residues in other cytoplasmic loops or insertion of cytochrome b562 into cytoplasmic loop VI/VII has no significant effect. The studies demonstrate that the last two cytoplasmic loops in lactose permease comprise a discontinuous epitope for monoclonal antibody 4B11 and thereby provide independent evidence for the conclusion that helices VIII-XI are in close proximity.

Antibodies, Monoclonal↗

PubMed1997

A cohort mortality study of construction workers.

This report presents a mortality study among the 17,344 members of the Construction Workers' Health Insurance Society of Mie Prefecture in Japan. The study period was between 1973 and 1993. During this period, 480 members died. Age-specific mortality rates of Mie Prefecture were used as comparison standards. Significantly elevated standardized mortality ratio (SMR) and proportionate mortality ratio (PMR) were observed for "accidents and adverse effects." In addition, the PMRs of all cancers and "cancers of trachea, bronchus and lung" were also significantly elevated. The job classifications were reorganized into three groups, according to the frequency of asbestos exposure the workers experienced on the construction sites. The asbestos exposure was based on job classifications among 7,411 workers who had completed a self-administered survey questionnaire. In the frequent-exposure group, the PMR was significantly elevated for all cancers. In the medium-exposure group, the SMRs were significantly elevated for all cancers and "cancer of trachea, bronchus and lung." The PMR was significantly elevated for "cancer of trachea, bronchus and lung." In the less-exposure, group, the PMR was significantly elevated for "accidents and adverse effects." This study provided support for the hypothesis that working in the construction industry might be associated with high risks for asbestos-associated cancers and accidental deaths.

Accidents, Occupational↗