A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR.

Skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta synthesized both apparently normal type I procollagen and a type I procollagen that had slow electrophoretic mobility because of posttranslational overmodifications. The thermal unfolding of the collagen molecules as assayed by protease digestion was about 2 degrees C lower than normal. It is surprising, however, that collagenase A and B fragments showed an essentially normal melting profile. Assay of cDNA heteroduplexes with a new technique involving carbodiimide modification indicated a mutation at about the codon for amino acid 550 of the alpha 1(I) chain. Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene. The results here confirm previous indications that the effects of glycine substitutions in type I procollagen are highly position specific. They also demonstrate that a recently described technique for detecting single-base differences by carbodiimide modification of DNA heteroduplexes can be effectively employed to locate mutations in large genes.     

Calcium-dependent protein kinase 29 modulates PIN-FORMED polarity and Arabidopsis development via its own phosphorylation code

PIN-FORMED (PIN)-mediated polar auxin transport (PAT) is involved in key developmental processes in plants. Various internal and external cues influence plant development via the modulation of intracellular PIN polarity and, thus, the direction of PAT, but the mechanisms underlying these processes remain largely unknown. PIN proteins harbor a hydrophilic loop (HL) that has important regulatory functions; here, we used the HL as bait in protein pulldown screening for modulators of intracellular PIN trafficking in Arabidopsis thaliana. Calcium-dependent protein kinase 29 (CPK29), a Ca²⁺-dependent protein kinase, was identified and shown to phosphorylate specific target residues on the PIN-HL that were not phosphorylated by other kinases. Furthermore, loss of CPK29 or mutations of the phospho-target residues in PIN-HLs significantly compromised intracellular PIN trafficking and polarity, causing defects in PIN-mediated auxin redistribution and biological processes such as lateral root formation, root twisting, hypocotyl gravitropism, phyllotaxis, and reproductive development. These findings indicate that CPK29 directly interprets Ca²⁺ signals from internal and external triggers, resulting in the modulation of PIN trafficking and auxin responses.

Ca²⁺-dependent protein kinase 29 directly phosphorylates the hydrophilic loop of PIN-FORMED proteins to modulate their intracellular trafficking and Arabidopsis development.     

Hypertension,calcium channel and pyridoxine (vitamin B6)

The moderately pyridoxine (vitamin B₆)-deficient male rat was introduced by us as an animal model (B6DHT) for the study of hypertension. Hypertension in this rat is associated with increased sympathetic stimulation. Arterial segments from B6DHT rats maintained a higher resting tone. The influx of ⁴⁵calcium into intracellular compartment of the vascular smooth muscle of the caudate artery of B6DHT rats was also enhanced. Administration of pyridoxine attenuated the hypertension in B6DHT rats as well as in genetic or dietary-induced moderately hypertensive conditions such as in the Zucker obese rat and sucrose or low calcium-fed rats. However, pyridoxine did not have any effect or the spontaneously hypertensive rat. All classes of calcium channel blockers were effective in lowering the systolic blood pressure of B6DHT rats. The increased in vitro influx of⁴⁵ calcium into intracellular compartment of artery segments of B6DHT rats as well as the BAY K 8644-induced influx of⁴⁵ calcium into artery segments from normal rats were blocked by pyridoxal phosphate as well as by dihydropyridine-sensitive calcium channel blockers (DHP). Pyridoxal phosphate (PLP) in vitro enhances the binding of calcium channel antagonists to membrane preparations from vascular tissue. PLP corrects the membrane abnormality in responsive hypertensive conditions and thus, could be an endogenous modulator of DHP - sensitive calcium channels.     

Studies on metabolism of vitamin A. The effect of hormones on gestation in retinoate-fed female rats

1. The preparation of cell suspensions by treatment of chick embryo hearts with collagenase at various stages of development is described. 2. Measurements of oxygen consumption, incorporation of labelled leucine into protein and accumulation of labelled alpha-aminoisobutyric acid against a concentration gradient indicated a long-lasting viability of the isolated heart cells in vitro; a satisfactory preservation of subcellular structures, including plasma membrane, was assessed by electron-microscopic examination. 3. The rate of alpha-aminoisobutyric acid accumulation by cardiac cells isolated from hearts at different stages of embryological development decreased with aging; insulin stimulated the intracellular accumulation of this amino acid analogue. 4. Insulin increased the uptake by isolated heart cells of several (14)C-labelled naturally occurring amino acids; however, the fraction of amino acid taken up by the cells that was recovered free intracellularly, and therefore the concentration ratio (between intracellular water and medium), was enhanced by the hormone only with glycine, proline, serine, threonine, histidine and methionine. When isolated heart cells were incubated in the presence of a mixture of labelled amino acids, the addition of insulin increased the disappearance of radioactivity from the medium. 5. The general pattern of amino acid transport (in the absence and in the presence of insulin) in isolated cardiac cells was similar to that found in intact hearts, suggesting that the biological preparation described in this paper might be useful for studies of cell permeability and insulin action.     

A unique Ni2+ ‐dependent and methylglyoxal‐inducible rice glyoxalase I possesses a single active site and functions in abiotic stress response

The glyoxalase system constitutes the major pathway for the detoxification of metabolically produced cytotoxin methylglyoxal (MG) into a non‐toxic metabolite d ‐lactate. Glyoxalase I (GLY I) is an evolutionarily conserved metalloenzyme requiring divalent metal ions for its activity: Zn²⁺ in the case of eukaryotes or Ni²⁺ for enzymes of prokaryotic origin. Plant GLY I proteins are part of a multimember family; however, not much is known about their physiological function, structure and metal dependency. In this study, we report a unique GLY I (OsGLYI‐11.2) from Oryza sativa (rice) that requires Ni²⁺ for its activity. Its biochemical, structural and functional characterization revealed it to be a monomeric enzyme, possessing a single Ni²⁺ coordination site despite containing two GLY I domains. The requirement of Ni²⁺ as a cofactor by an enzyme involved in cellular detoxification suggests an essential role for this otherwise toxic heavy metal in the stress response. Intriguingly, the expression of OsGLYI‐11.2 was found to be highly substrate inducible, suggesting an important mode of regulation for its cellular levels. Heterologous expression of OsGLYI‐11.2 in Escherichia coli and model plant Nicotiana tabacum (tobacco) resulted in improved adaptation to various abiotic stresses caused by increased scavenging of MG, lower Na⁺/K⁺ ratio and maintenance of reduced glutathione levels. Together, our results suggest interesting links between MG cellular levels, its detoxification by GLY I, and Ni²⁺ – the heavy metal cofactor of OsGLYI‐11.2, in relation to stress response and adaptation in plants.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis.     

Mycobacterium tuberculosis secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide-induced NF-kappaB transactivation by downregulation of reactive oxidative species (ROS) production

Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people annually and is considered one of the most successful intracellular pathogens to persist inside the host macrophage. Recent studies have implicated the role of RD-1 region of Mtb genome in the mycobacterial pathogenesis. The role of RD-1-encoded secretory proteins of Mtb in modulation of macrophage function has not been investigated in detail. Here we show that RD-1 encoded two major secretory proteins, namely, culture filtrate protein-10 kDa (CFP-10) and early secreted antigenic target-6 kDa (ESAT-6), and their 1:1 CFP-10:ESAT6 complex inhibit production of reactive oxidative species (ROS) in RAW264.7 cells. These proteins also downregulated the bacterial lipopolysaccharide (LPS)-induced ROS production, which, in turn, downregulated LPS-induced nuclear factor-kappaB (NF-kappaB) p65 DNA-binding activity, as well as inhibited the NF-kappaB-dependent reporter gene (chloramphenicol acetyl transferase) expression in the treated macrophages. Moreover, addition of N-acetyl cysteine, which is a scavenger of ROS, also inhibited LPS-induced reporter gene expression by scavenging the ROS, thereby preventing NF-kappaB transactivation. These studies indicate that the secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex of Mtb can inhibit LPS-induced NF-kappaB-dependent gene expression via downregulation of ROS production.