Interleukin-8 activates microtubule-associated protein 2 kinase (ERK1) in human neutrophils

The signal transduction initiated by the human cytokine interleukin-8 (IL-8), the main chemotactic cytokine for neutrophils, was investigated and found to encompass the stimulation of protein kinases. More specifically, IL-8 caused a transient, dose and time dependent activation of a Ser/Thr kinase activity towards myelin basic protein (MBP) and the MBP-derived peptide APRTPGGRR patterned after the specific concensus sequence in MBP for ERK enzymes. The activated MBP kinase was furthermore identified as an extracellular signal regulated kinase (ERK1) based on several criteria such as substrate specificity, molecular weight, activation-dependent mobility shift, and recognition by anti-ERK antibodies. For comparison, the chemotactic response of neutrophils to a stimulus of bacterial origin (fMet-Leu-Phe or fMLP) was also examined and found to involve the activation of a similar ERK enzyme. The present data clearly indicate that in terminally differentiated, non-proliferating human cells, the MBP kinase/ERK activity can serve other purposes than mitogenic signaling, and that processes such as chemotaxis, induced by bacterial peptides as well as by human cytokines like IL-8, involve the regulation of ERK enzyme.Abbreviations IL-8 interleukin-8 - fMLP fMet-Leu-Phe - MBP myelin basic protein - ERK extracellular signal regulated kinase - MAP2 microtubule-associated protein 2 - PK-A cAMP dependent protein kinase - PKI protein kinase inhibitor - PMSF phenyl-methanesulfonyl fluoride - PVDF poly-vinylidene difluoride - HBSF Hank's buffered salt solution - DAB 3,3-diaminobenzidine tetrahydrochloride - PNPP p-nitrophenyl-phosphate - HSA human serum albumin - EGTA [ethylenebis (oxyethylenenitrilo)]tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Cardiovascular and metabolic responses to noradrenaline in men acclimatized to cold baths

The purpose of this study was to see whether artificial acclimatization to cold would reduce the pressor response to noradrenaline (NA) as natural acclimatization has been shown to do, and whether it would induce nonshivering thermogenesis. Three white men were infused with NA at four dosage levels between 0.038 and 0.300 g·kg^–1·min^–1 (2–23 g·min^–1), before and after artificial acclimatization to cold and again 4 months later when acclimatization had decayed. Acclimatization was induced by ten daily cold (15°Q baths of 30–60 min followed by rapid rewarming in hot (38–42°C) water, and was confirmed by tests of the subjects responses to whole-body cooling in air. Three control subjects also underwent the first and third tests. Acclimatization substantially reduced the pressor response to NA at 0.150 and 0.300 g·kg^–1·min^–1, confirming earlier findings by the same technique in naturally acclimatized men, and its decay increased this response to beyond its initial levels (P<0.05 for both changes). Acclimatization did not change the response to NA of heart rate, subjective impressions, skin temperature of finger and toe, pulmonary ventilation, or plasma free fatty acids and ketone bodies. At no time did NA increase oxygen consumption, or increase skin temperature or heat flow over reported sites of brown fat. These findings would seem to show that acclimatization to cold reduces sensitivity to the pressor effect of NA but does not induce nonshivering thermogenesis, and that the reduced sensitivity is replaced by a hypersensitivity to NA when acclimatization decays.     

Human bone marrow stromal cells express an osteoblastic phenotype in culture

Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [³H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D₃ by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with⁴⁵Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.     

Induction of the human growth hormone gene placed under human hsp70 promoter control in mouse cells: A quantitative indicator of metal toxicity

An in vitro test method for general metal toxicity screening was designed, based on the cellular response to stress. The expression of a transfected human growth hormone gene sequence driven by the human heat-shock protein 70 promoter in NIH/3T3 cells was used as marker of noxious contact with metal compounds. Out of a series of31 metals, 17 were competentfor inducing this stress response system. According to the effective concentration and to the intensity of the response, three different clusters of positive compounds emerged and were ranked as strong, intermediate strength and weak inducers. These results correlated well with data from other in vivo and in vitro metal toxicity studies, including LD₅₀ in mice. Apparently the positivelnegative compounds also fitted well with data from genotoxicity and carcinogenesis studies on metal salts.Abbreviations hGH human growth hormone - hsp70 70 kDa heat-shock protein     

Fibronectin turnover in human mesangial cell cultures as affected by Adriamycin

Fibronectin (FN) turnover and turnover changes induced by the anticancer drug Adriamycin (ADR) were measured in human mesangial cells (HMC) in vitro. HMC cultures synthesize cellular FN (2.2+-0.3% of totalprotein synthesis; n = 12) which is secreted and incorporated into a fibrillar extracellular matrix (ECM). A 24 hr incubation of HMC with ADR (0.5–5 g/ml) resulted in an accumulation of FN in the culture medium, with a maximum increase following 5 pglml(7.3+-2.3pg/cell vs. controls: 4.4+-1.9pg/cell; n= 10). Correspondingly, radioactively labeled immunoprecipitable FN was increased in a dosage-dependent manner in the culture medium up to 50% vs. controls. The incorporation of radioactively labeled FN into ECM was significantly increased following 2 g ADR/ml. In accordance, immunofZuorescence staining revealed an expansion ofpericellular FNfibers in cultures exposed to 2 g ADR/ml. Concomitant with the accumulation of extracelhlar FN, radioactively labeled FN in the cells was reduced by 22%. Qualitative characterization of FN patterns revealed a diminished number of degradation products in the culture medium ofADR-treated HMC. These data suggest thatADR interferes with the turnover of FN secreted by HMC in vitro in such a way that FN accumulates extracellularly. This in turn leads to a reduced FN synthesis. These findings are compatible with a loss of urinary FN degradation products accompanying the onset ofproteinuria in ADR-treated rats.Abbreviations ADR adriamycin - BSA bovine serum albumin - DTT dithiothreitol - ECM extracellular matrix - EDTA ethylenediamine tetraacetic acid disodium salt - ELISA enzyme-linked immunosorbent assay - FCS fetal calf serum - FITC fluorescein isothiocyanate - FN fibronectin - HMC human mesangial cell - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Rapid backward movement of anaphase chromosome whose kinetochore fibers were cut by ultraviolet microbeam irradiation

Summary— kinetochore spindle fibers in meiosis I and II grasshopper spermatocytes were cut with a heterochromatic ultraviolet (UV) microbeam converging on the specimen to form a slit-shaped microspot 1.5 × 8 μm or 3 × 8 μm. A total exposure of 3 × 10^?8 joules per μm² was administered within 0.8–2.4 s, which was sufficient for severing. The cells were observed with a high extinction polarizing microscope or phase contrast optics and a record made by time-lapse video microscopy, continuously before, during and after the irradiation. When kinetochore fibers were irradiated i anaphase with UV, an area of reduced birefringence (ARB) was produced at the exposed site. The newly created + ends of the microtubules rapidly disassembled poleward, at a constant speed of 17 μm/min. The — ends at the edge of ARB also depolymerized at a slower rate. When a kinetochore fiber was cut with UV in early anaphase at which time its associated chromosome had not disjoined from the partner chromosome, the chromosome of the irradiated kinetochore fiber moved rapidly back to its partner. The speed during this movement was faster than the normal poleward chromosome movement in anaphase by an order to magnitude or more. When a kinetochore and its associated kinetochore fiber were included in the irradiation are, the effects were more pronounced than the effects of irradiation on a kinetochore fiber alone; the direction of the line connecting the irradiated half-bivalent with the partner half-bivalent deviated so much from the longitudinal axis of the original spindle with time that the division assumed a tripolar figure.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.