Altered processing of integrin receptors during keratinocyte activation

We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells.     

Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes

Adult T-cell leukemia (ATL)-related cells have the ability to produce a newly-isolated calcium-regulating protein, parathyroid hormone-related protein (PTHrP). The present study revealed that lectin-stimulated normal lymphocytes produce immunoreactive (IR)-PTHrP. When the T-cell-enriched fraction was purified from normal lymphocytes and then treated with lectin, a similar amount of IR-PTHrP was detected, suggesting that IR-PTHrP is an actual product of T-lymphocytes. A biologically active fragment of PTHrP, PTHrP(1-34), suppressed DNA synthesis in lectin-stimulated lymphocytes at concentrations greater than 50 pg/mL; the same concentration range of IR-PTHrP detected in the cultured media of lectin-stimulated lymphocytes. Therefore, it is reasonable to postulate that PTHrP is a cytokine inhibiting the cellular growth of normal lymphocytes.     

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli

The product of the malE—lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Echerichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827–2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.     

Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.     

Direct binding of Toll-like receptor 2 to zymosan,and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-alpha secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-kappaB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-alpha secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.     

Age-related effects of major and trace element concentrations in rat liver and their mutual relationships

The concentrations of 22 major and trace elements in livers from rats aging from 5 to 113 weeks old were determined. The rats investigated were the same rats previously reported with respect to 29 elements in bones (femur) and 26 elements in kidneys. The samples were decomposed with high-purity nitric acid and hydrogen peroxide. Seven elements (Na, Mg, P, K, Ca, Fe and Zn) were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), and 15 elements (Mn, Co, Cu, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Cs, Ba, Pb and Bi) were determined by inductively coupled plasma mass spectrometry (ICP-MS). Analysis of variance (ANOVA) for age variations indicated that the concentrations of many elements, such as Mg, P, K, Mn, Fe, Cu, Zn, Sr, Mo and Cd, were almost constant across the ages of the rats with the exception of 5 weeks old (p > 0.05). Arsenic, Pb and Bi showed significant increasing trends, while Na and Co showed decreasing trends (p < 0.01). Selenium showed a decreasing trend except at the initial stage of 5–9 weeks old. Calcium, Rb, Sn, Sb, Cs and Ba showed significant age-related variations, but their patterns were not monotonic. The liver clearly contrasts with the kidneys, in which many elements showed significant age-related variations with increasing trends. The concentration ranges of Mg, P, K, Mn, Cu, Zn, and Mo were controlled within 15% across all ages of rats. The homeostasis of the aforementioned elements may be well established in the liver. The toxic elements, such as Cd, Pb and Bi, showed a narrow concentration range among age-matched rats.     

Development of simple sequence repeat markers and construction of a high-density linkage map of Capsicum annuum

To facilitate marker-assisted breeding and genetic analyses of pepper (Capsicum annuum), we developed non-redundant 2- or 3-base simple sequence repeat (SSR) markers from enriched C. annuum genomic libraries and from C. annuum cDNA sequences in public databases. The SSR-enriched libraries were constructed using combinations of three restriction enzymes (AluI, HaeIII, and RsaI) and two biotinylated oligonucleotides [b(GA)₁₅ and b(CA)₁₅]. Ultimately, we obtained 1,736 genomic SSR markers and 1,344 cDNA-derived SSR markers from 6,528 clones and 13,003 sequences, respectively. We mapped 597 markers, including 265 of the newly developed SSR markers, onto a linkage map by using doubled-haploid (DH) lines derived from an intraspecific cross of two pure lines of C. annuum (K9-11 × MZC-180). The map, designated as the KL-DH map, consisted of 12 linkage groups. The map covered a genetic distance of 2,028 cM, and the average distance between markers was less than 4 cM. The frame structure of the KL-DH map was compared with the published standard conserved ortholog set II (COSII) map, which was derived from an interspecific F₂ population (C. frutescens × C. annuum), by using tomato (Solanum lycopersicum) chromosomal sequences to bridge the two maps. The intraspecific KL-DH map constructed in this study and the interspecific COSII map were similar in map length and marker distribution, suggesting that the KL-DH map covers nearly the whole genome of C. annuum.