Roscovitine blocks collecting duct cyst growth in Cep164-deficient kidneys.

Nephronophthisis is an autosomal recessive kidney disease with high genetic heterogeneity. Understanding the functions of the individual genes contributing to this disease is critical for delineating the pathomechanisms of this disorder. Here, we investigated kidney function of a novel gene associated with nephronophthisis, CEP164, coding a centriolar distal appendage protein, using a Cep164 knockout mouse model. Collecting duct-specific deletion of Cep164 abolished primary cilia from the collecting duct epithelium and led to rapid postnatal cyst growth in the kidneys. Cell cycle and biochemical studies revealed that tubular hyperproliferation is the primary mechanism that drives cystogenesis in the kidneys of these mice. Administration of roscovitine, a cell cycle inhibitor, blocked cyst growth in the cortical collecting ducts and preserved kidney parenchyma in Cep164 knockout mice. Thus, our findings provide evidence that therapeutic modulation of cell cycle activity can be an effective approach to prevent cyst progression in the kidney.

Preclinical antitumor activity of cabazitaxel, a semisynthetic taxane active in taxane-resistant tumors.

PURPOSE: Taxanes are important chemotherapeutic agents with proven efficacy in human cancers, but their use is limited by resistance development. We report here the preclinical characteristics of cabazitaxel (XRP6258), a semisynthetic taxane developed to overcome taxane resistance. EXPERIMENTAL DESIGN: Cabazitaxel effects on purified tubulin and on taxane-sensitive or chemotherapy-resistant tumor cells were evaluated in vitro. Antitumor activity and pharmacokinetics of intravenously administered cabazitaxel were assessed in tumor-bearing mice. RESULTS: In vitro, cabazitaxel stabilized microtubules as effectively as docetaxel but was 10-fold more potent than docetaxel in chemotherapy-resistant tumor cells (IC50 ranges: cabazitaxel, 0.013-0.414 µmol/L; docetaxel, 0.17-4.01 µmol/L). The active concentrations of cabazitaxel in these cell lines were achieved easily and maintained for up to 96 hours in the tumors of mice bearing MA16/C tumors treated with cabazitaxel at 40 mg/kg. Cabazitaxel exhibited antitumor efficacy in a broad spectrum of murine and human tumors (melanoma B16, colon C51, C38, HCT 116, and HT-29, mammary MA17/A and MA16/C, pancreas P03 and MIA PaCa-2, prostate DU 145, lung A549 and NCI-H460, gastric N87, head and neck SR475, and kidney Caki-1). Of particular note, cabazitaxel was active in tumors poorly sensitive or innately resistant to docetaxel (Lewis lung, pancreas P02, colon HCT-8, gastric GXF-209, mammary UISO BCA-1) or with acquired docetaxel resistance (melanoma B16/TXT). CONCLUSIONS: Cabazitaxel is as active as docetaxel in docetaxel-sensitive tumor models but is more potent than docetaxel in tumor models with innate or acquired resistance to taxanes and other chemotherapies. These studies were the basis for subsequent clinical evaluation.

Selenium preserves mitochondrial function, stimulates mitochondrial biogenesis, and reduces infarct volume after focal cerebral ischemia.

BACKGROUND: Mitochondrial dysfunction is one of the major events responsible for activation of neuronal cell death pathways during cerebral ischemia. Trace element selenium has been shown to protect neurons in various diseases conditions. Present study is conducted to demonstrate that selenium preserves mitochondrial functional performance, activates mitochondrial biogenesis and prevents hypoxic/ischemic cell damage. RESULTS: The study conducted on HT22 cells exposed to glutamate or hypoxia and mice subjected to 60-min focal cerebral ischemia revealed that selenium (100 nM) pretreatment (24 h) significantly attenuated cell death induced by either glutamate toxicity or hypoxia. The protective effects were associated with reduction of glutamate and hypoxia-induced ROS production and alleviation of hypoxia-induced suppression of mitochondrial respiratory complex activities. The animal studies demonstrated that selenite pretreatment (0.2 mg/kg i.p. once a day for 7 days) ameliorated cerebral infarct volume and reduced DNA oxidation. Furthermore, selenite increased protein levels of peroxisome proliferator-activated receptor-γ coactivator 1alpha (PGC-1α) and nuclear respiratory factor 1 (NRF1), two key nuclear factors that regulate mitochondrial biogenesis. Finally, selenite normalized the ischemia-induced activation of Beclin 1 and microtubule-associated protein 1 light chain 3-II (LC3-II), markers for autophagy. CONCLUSIONS: These results suggest that selenium protects neurons against hypoxic/ischemic damage by reducing oxidative stress, restoring mitochondrial functional activities and stimulating mitochondrial biogenesis.

Anti-microtubule activity of tubeimoside I and its colchicine binding site of tubulin.

BACKGROUND: Tubeimoside I (TBMS1) was isolated from the tubers of Bolbostemma paniculatum (Maxim.) Franquet. TBMS1 shows potent anti-tumor activity. The present study was conducted to investigate the anti-microtubule role of TBMS1 and its binding site of tubulin. METHODS: Cell growth inhibition was measured by MTT after treatment with TBMS1. Uptake kinetics of TBMS1 by human nasopharyngeal carcinoma CNE-2Z cell line (CNE-2Z) was assayed by HPLC. Microtubule protein (MTP) was prepared from porcine brain through two cycles of polymerization-depolymerization in a high molarity buffer. Inhibition of MTP polymerization induced by TBMS1 was determined by a turbidity measurement and a sedimentation assay; the interactions of TBMS1 with tubulin within CNE-2Z cells were investigated by immunofluorescence microscopy and immunoblotting. TBMS1 was tested for its ability to inhibit binding of known tubulin ligands through competitive binding assay. RESULTS: TBMS1 displayed growth inhibitory activity against CNE-2Z cells with IC(50) value of 16.7 microM for 72 h. HPLC analysis of TBMS1 uptake by CNE-2Z cells displayed the initial slow TBMS1 uptake and then gradually reaching an maximum uptake near 18 h. CNE-2Z cells treated with TBMS1 (25 microM, 3 h) were sufficient to cause the microtubular network disruption. Immunoblot analysis showed that the proportion of cytosolic tubulin of cells treated with TBMS1 increased in a time- and concentration-dependent manner. TBMS1 did not inhibit the binding of vinblastine to tubulin. Colchicine binding to tubulin was inhibited in the presence of TBMS1. CONCLUSIONS: TBMS1 is an anti-microtubule agent, and its binding site of tubulin is the colchicine binding site of tubulin.

Upregulation of activating transcription factor 3 (ATF3) by intrinsic CNS neurons regenerating axons into peripheral nerve grafts.

The expression of the transcription factor ATF3 in the brain was examined by immunohistochemistry during axonal regeneration induced by the implantation of pieces of peripheral nerve into the thalamus of adult rats. After 3 days, ATF3 immunoreactivity was present in many cells within approximately 500 mum of the graft. In addition, ATF3-positive cell nuclei were found in the thalamic reticular nucleus (TRN) and medial geniculate nuclear complex (MGN), from which most regenerating axons originate. CNS cells with ATF3-positive nuclei were predominantly neurons and did not show signs of apoptosis. The number of ATF3-positive cells had declined by 7 days and further by 1 month after grafting when most ATF3-positive cells were found in the TRN and MGN. 14 days or more after grafting, some ATF3-positive nuclei were distorted and may have been apoptotic. In some experiments of 1 month duration, neurons which had regenerated axons to the distal ends of grafts were retrogradely labeled with DiAsp. ATF3-positive neurons in these animals were located in regions of the TRN and MGN containing retrogradely labeled neurons and the great majority were also labeled with DiAsp. SCG10 and c-Jun were found in neurons in the same regions as retrogradely labeled and ATF3-positive cells. Thus, ATF3 is transiently upregulated by injured CNS neurons, but prolonged expression is part of the pattern of gene expression associated with axonal regeneration. The co-expression of ATF3 with c-jun suggests that interactions between these transcription factors may be important for controlling the program of gene expression necessary for regeneration.

A tektin homologue is decreased in chlamydomonas mutants lacking an axonemal inner-arm dynein.

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found approximately 80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a-g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment.

A sesquiterpene lactone, costunolide, interacts with microtubule protein and inhibits the growth of MCF-7 cells.

Costunolide is an active sesquiterpene lactone of medicinal herbs with anti-inflammatory and potential anti-cancer activity. Nevertheless, the pharmacological pathways of costunolide have not yet been fully elucidated. In this study we showed that costunolide exerts a dose-dependent antiproliferative activity in the human breast cancer MCF-7 cells. In addition, light microscopy observations indicated that costunolide affected nuclear organization and reorganized microtubule architecture. The antiproliferative and antimicrotubular effects of costunolide were not influenced by paclitaxel, well-known microtubule-stabilizing anticancer agent. The microtubule-interacting activity of costunolide was confirmed by in vitro studies on purified microtubular protein. In fact, costunolide demonstrated polymerizing ability, by inducing the formation of well organized microtubule polymers. Our data suggest an interaction of costunolide with microtubules, which may represent a new intracellular target for this drug.

Transcriptional upregulation of SCG10 and CAP-23 is correlated with regeneration of the axons of peripheral and central neurons in vivo.

We have compared SCG10 and CAP-23 expression with that of GAP-43 during axonal regeneration in the peripheral and central nervous systems (PNS, CNS) of adult rats. SCG10, CAP-23, and GAP-43 mRNAs were strongly upregulated by motor and dorsal root ganglion (DRG) neurons following sciatic nerve crush, but not after dorsal rhizotomy. When the sciatic nerve was cut and ligated to prevent reinnervation of targets, expression of all three mRNAs was prolonged. Neurons in the thalamic reticular nucleus and deep cerebellar nuclei transiently upregulated these mRNAs after axotomy, and showed prolonged upregulation of all three molecules when regenerating axons into peripheral nerve grafts inserted into the thalamus of cerebellum. Neurons in the dorsal thalamus and cerebellar cortex showed poor regenerative capacity and most did not upregulate any of these mRNAs. Thus, in both PNS and CNS neurons, the transcription of SCG10, CAP-23, and GAP-43 mRNAs is coregulated following axotomy and during regeneration. Signals from living peripheral nerve appear to maintain expression of all three mRNAs in regenerating neurons, and in PNS neurons downregulation correlates with target reinnervation. Thus, SCG10 and CAP-23, as well as GAP-43, are likely to be important neuronal determinants of regenerative ability.

Expression pattern of dd4, a sole member of the d4 family of transcription factors in Drosophila melanogaster.

In vertebrates, three members of the d4 gene family code for proteins, which are believed to function as transcription factors and involved in regulation of various intracellular processes. One member of the family, ubi-d4/requiem is ubiquitously expressed gene and two other, neuro-d4 and cer-d4, are expressed predominantly in the neural tissues (Nucleic Acids Res. 20 (1992) 5579; Biochim. Biophys. Acta 14 (1992) 172; Mamm. Genome 11 (2000) 72; Mamm. Genome 12 (2001) 862). Typically, d4 proteins show distinct domain organisation with domain 2/3 in the N-terminal, Krüppel-type zinc finger in the central and two adjacent PHD-fingers (d4-domain) in the C-terminal part of the molecule. However, alternative splicing, which is responsible for complex expression patterns of both neurospecific members of the family, generates multiple protein isoforms lacking certain domains (Nucleic Acids Res. 20 (1992) 5579; Genomics 36 (1996) 174; Mamm. Genome 11 (2000) 72; Mamm. Genome 12 (2001) 862). Exact function of d4 proteins is unclear but their involvement in regulation of differentiation and apoptotic cell death has been proposed (J. Biol. Chem. 269 (1994) 29515; Mamm. Genome 11 (2000) 72; Mamm. Genome 12 (2001) 862). Here we identified a single gene, dd4, in the genome of Drosophila melanogaster, the protein product of which could be assigned to the d4 family. Expression of dd4 is regulated during Drosophila development, and is most prominent in syncytial embryos and later in the embryonic nervous and reproductive systems. In flies dd4 mRNA is found in most tissues but the highest level of expression is detected in ovaries.

Regulation and subcellular localization of the microtubule-destabilizing stathmin family phosphoproteins in cortical neurons.

Stathmin is a ubiquitous cytosolic phosphoprotein, preferentially expressed in the nervous system, and the generic element of a protein family that includes the neural-specific proteins SCG10, SCLIP, and RB3 and its splice variants, RB3' and RB3". All phosphoproteins of the family share with stathmin its tubulin binding and microtubule (MT)-destabilizing activities. To understand better the specific roles of these proteins in neuronal cells, we performed a comparative study of their expression, regulation, and intracellular distribution in embryonic cortical neurons in culture. We found that stathmin is highly expressed ( approximately 0.25% of total proteins) and uniformly present in the various neuronal compartments (cell body, dendrites, axon, growth cones). It appeared mainly unphosphorylated or weakly phosphorylated on one site, and antisera to specific phosphorylated sites (serines 16, 25, or 38) did not reveal a differential regulation of its phosphorylation among neuronal cell compartments. However, they revealed a subpopulation of cells in which stathmin was highly phosphorylated on serine 16, possibly by CaM kinase II also active in a similar subpopulation. The other proteins of the stathmin family are expressed about 100-fold less than stathmin in partially distinct neuronal populations, RB3 being detected in only about 20% of neurons in culture. In contrast to stathmin, they are each mostly concentrated at the Golgi apparatus and are also present along dendrites and axons, including growth cones. Altogether, our results suggest that the different members of the stathmin family have complementary, at least partially distinct functions in neuronal cell regulation, in particular in relation to MT dynamics.

Celogentins A-C, new antimitotic bicyclic peptides from the seeds of Celosia argentea.

Three new bicyclic peptides, celogentins A (1), B (2), and C (3), have been isolated together with a known-related peptide, moroidin (4), from the seeds of Celosia argentea, and their structures including absolute stereochemistry were determined by using extensive NMR methods and chemical means. Celogentins A (1), B (2), and C (3) inhibited the polymerization of tubulin, and celogentin C (3) was four times more potent than moroidin (4) in the inhibitory activity. Structure-activity relationship study using moroidin derivatives 5-7 and analogue 8 as well as celogentins A-C (1-3) and moroidin (4) indicates that the bicyclic ring system including unusual non-peptide connections among beta(s)-Leu, Trp, and His residues characteristic of celogentins and moroidin, with ring size and conformations suitable for interaction with tubulin would be important for their biological activity.

Stathmin family proteins display specific molecular and tubulin binding properties.

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3'/RB3") are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific "stathmin-like domain" and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two alpha/beta tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3'. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.

New mutations in MID1 provide support for loss of function as the cause of X-linked Opitz syndrome.

Opitz syndrome (OS) is a genetically heterogeneous malformation disorder. Patients with OS may present with a variable array of malformations that are indicative of a disturbance of the primary midline developmental field. Mutations in the C-terminal half of MID1, an RBCC (RING, B-box and coiled-coil) protein, have recently been shown to underlie the X-linked form of OS. Here we show that the MID1 gene spans at least 400 kb, almost twice the distance originally reported and has a minimum of six mRNA isoforms as a result of the alternative use of 5' untranslated exons. In addition, our detailed mutational analysis of MID1 in a cohort of 15 patients with OS has resulted in the identification of seven novel mutations, two of which disrupt the N-terminus of the protein. The most severe of these (E115X) is predicted to truncate the protein before the B-box motifs. In a separate patient, a missense change (L626P) was found that also represents the most C-terminal alteration reported to date. As noted with other C-terminal mutations, GFP fusion constructs demonstrated that the L626P mutant formed cytoplasmic clumps in contrast to the microtubular distribution seen with the wild-type sequence. Notably, however, both N-terminal mutants showed no evidence of cytoplasmic aggregation, inferring that this feature is not pathognomonic for X-linked OS. These new data and the finding of linkage to MID1 in the absence of a demonstrable open reading frame mutation in a further family support the conclusion that X-linked OS results from loss of function of MID1.

Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas.

A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca(2+)-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca(2+)-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca(2+)/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca(2+)-activated, Ca(2+)-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.

The upregulation of messenger ribonucleic acids during 17alpha, 20beta-dihydroxy-4-pregnen-3-one-induced ovulation in the perch ovary.

While progestins appear to be involved in the local ovarian regulation of vertebrate ovulation, their specific role is unclear. In yellow perch (Perca flavescens) the progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20-P), stimulates ovulation in vitro and this induction requires gene activation. Therefore, the perch model was used to isolate progestin-upregulated mRNAs. Perch ovaries were incubated for 32 h with or without 17,20-P (0.1 microg/ml). Messenger ribonucleic acids were isolated from the tissue and used for differential display PCR (DDPCR). From DDPCR, 5 bands were eventually obtained that were verified by Northern analysis to be consistently upregulated by 17,20-P at 32 h. Using these bands, full-length cDNAs were obtained by library screening and completely sequenced. Based on similarity to known sequences, four of the cDNAs presumably encode for perch forms of (1) neprilysin (PNEP-1; 63% identical); (2) a lysyl oxidase-type protein (PLO-2; 43.2% identical); (3) calmodulin (PCAL-1; 100% identical); and (4) a microtubule aggregate-like protein (PMAP-1; 29.6% identical). The fifth cDNA obtained from DDPCR most likely encodes for an egg protein and will be reported separately. Each of the cDNAs was used to probe Northern blots of ovarian mRNA taken at 0, 12, 24, 32 and 42 h of incubation with 17,20-P. This temporal Northern analysis verified that all four were upregulated by 32 h. In addition, PNEP and PMAP transcripts began to increase by 12 h, while PCAL and PLO transcripts remained elevated through 42 h. On Northern blots of RNA from other perch tissues, calmodulin was found in all tissues, PLO mRNA was ovarian specific, and PMAP mRNA was also present in the gills and liver. Multiple transcripts were observed for PNEP, but the ovarian form induced by 17,20-P was only found in high abundance in the heart. To our knowledge, this is the first report that specifically characterizes progestin upregulated mRNAs in the vertebrate ovary at ovulation.

MID2, a homologue of the Opitz syndrome gene MID1: similarities in subcellular localization and differences in expression during development.

The B-box family is an expanding new family of genes encoding proteins involved in diverse cellular functions such as developmental patterning and oncogenesis. A member of this protein family, MID1, is the gene responsible for the X-linked form of Opitz G/BBB syndrome, a developmental disorder characterized by defects of the midline structures. We now report the identification of MID2, a new transcript closely related to MID1. MID2 maps to Xq22 in human and to the syntenic region on the mouse X chromosome. The two X-linked genes share the same domains, the same exon-intron organization, a high degree of similarity at the protein level and the same subcellular localization, both being confined to the cytoplasm in association to micro-tubular structures. The expression pattern studied by RNA in situ hybridization in mouse revealed that Mid2 is expressed early in development and the highest level of expression is detected in the heart, unlike Mid1 for which no expression was detected in the developing heart. Together, these data suggest that midin and MID2 have a similar biochemical function but a different physiological role during development.

Differential regulation of the growth-associated proteins, GAP-43 and SCG-10, in response to unilateral cortical ablation in adult rats.

Synapse replacement after brain injury has been widely documented by anatomical studies in various parts of both the developing and adult nervous system. However, the molecular events that define the specificity of the empirically derived rules of reactive synaptogenesis in different regions of the adult brain remain unclear. In this study we examined the differential regulation of the lesion-induced response of the two growth-associated proteins, superior cervical ganglia-10 and growth-associated protein-43, after unilateral cortex ablation, and determined a hierarchical order for the lesion response from remaining afferent projection neurons originating from the contralateral cortex, ipsilateral thalamus and substantia nigra. We report that in response to unilateral cortex ablation both messenger RNA, by northern blot, and protein, by western blot, for superior cervical ganglia-10 but not growth-associated protein-43 was increased in the homologous area of the contralateral cortex but not the ipsilateral thalamus or substantia nigra. In addition, the specificity of the superior cervical ganglia-10 response, assessed by combined in situ hybridization and retrograde FluoroGold labeling of striatal afferent neurons, found that superior cervical ganglia-10 messenger RNA was increased prominently in layer V pyramidal neurons of the contralateral corticostriatal pathway but was unchanged in afferent projection neurons from the thalamus and substantia nigra. Furthermore, the increase in both superior cervical ganglia-10 messenger RNA and protein seen at three days postlesion in contralateral corticostriatal neurons coincides in time with the initiation of neurite outgrowth in the deafferented striatum by contralateral corticostriatal axons described in our previous ultrastructural study. However, if cortical input to the striatum was removed bilaterally the lesion-induced response for superior cervical ganglia-10 messenger RNA shifted secondarily to thalamostriatal neurons in the ipsilateral thalamus. These data provide evidence that superior cervical ganglia-10 and growth-associated protein-43 are differentially regulated in neurons of the contralateral corticostriatal pathway in response to unilateral cortex ablation and suggests that superior cervical ganglia-10 plays a role in the regulation of neurite outgrowth in the adult striatum after brain injury. However, the specific role that superior cervical ganglia-10 may play in reactive synaptogenesis remains unclear. In addition, our data suggest that a hierarchical order exists for the reinnervation of deafferented striatal neurons after unilateral cortex ablation with preference given to homologous axons from the contralateral cortex.

High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234.

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.

Posttranslational arginylation of soluble rat brain proteins after whole body hyperthermia.

We have previously reported the posttranslational addition of [14C]-arginine in the N-terminus of several soluble rat brain proteins. One of these proteins was identified as the microtubule-associated protein, the stable tubule only polypeptide (STOP). However, despite the fact that the biological significance of arginylation is not completely understood, some evidence associates it with proteolysis via the ubiquitin pathway. Since this degradative via is exacerbated as a response to stress, we studied in vitro the posttranslational [14C]-arginylation of cytosolic brain proteins of rats subjected to hyperthermia in vivo. Immediately after subjecting the animals to hyperthermia, a minor reduction (16%) in the acceptor capacity of [14C]-arginine into proteins was observed in comparison with animals maintained at 28 degrees C. However, in the animals allowed to recover for 3 h, an increase (46%) in the arginylation was observed concomitantly with a significant accumulation of the heat shock protein (70 kDa; hsp 70) when compared to the control animals. These findings suggest that the posttranslational arginylation of proteins participate in the heat shock response. The STOP protein of the soluble brain fraction of control animals, which in Western blot appears as a doublet band (125 and 130 kDa, respectively), is seen, after the hyperthermic treatment, as a single band of 125 kDa. The amount of 125 kDa protein, as well as the in vitro incorporation of [14C]-arginine, increases after hyperthermia in comparison with control animals. Following hyperthermic treatment, we observed a decrease in the amount of in vivo [35S]-methionine-labeled brain proteins. We speculate that, as observed for STOP protein, the increase in the degradation of protein that occurs in hyperthermia, would produce an increase in the amount of arginine acceptor proteins.

Stathmin is involved in S100A4-mediated regulation of cell cycle progression.

S100A4 is a cell proliferation- and cancer metastasis-related gene. Previous studies have shown that over-expression of S100A4 drives the cells into the S-phase of the cell cycle, with concomitant enhancement of p53 detection. This has led to the postulate that S100A4 could be controlling cell cycle progression by sequestering p53 and abrogating its G1-S checkpoint control. Cells induced by S100A4 to enter the S-phase do successfully negotiate the G2-M checkpoint control. Here we show that S100A4 is also involved in the regulation of control at this checkpoint. Stathmin is known to be associated, together with p53 in controlling G2-M transition. We present evidence that the expression of S100A4 and stathmin genes is up regulated in exponentially growing HeLa cells. They are down regulated in parallel when cell proliferation is inhibited by hyperthermia and 4-hydroxynonenal (4-HNE). We postulate that S100A4 might directly induce stathmin up regulation to enable cells to enter into mitosis. Since wild-type p53 is known to down regulate stathmin expression, we further postulate this might also involve S100A4-mediated sequestration of p53. The expression of heme oxygenase (HO-1), a stress-response protein, has been used to monitor effects of hyperthermia, 12-O-tetradecanoly phorbol 13-acetate (TPA) and 4-HNE. All these treatments induced HO-1 and also when cells growing in serum-deficiency were restored with full serum. HO-1 induction occurred irrespective of S100A4 expression status. HO-1 gene has responsive elements for many angiogenic agents and induces marked neovascularisation of tumours. We suggest therefore that S100A4 may not possess angiogenic properties.