Notch Is a Critical Component of the Mouse Somitogenesis Oscillator and Is Essential for the Formation of the Somites

Segmentation of the vertebrate body axis is initiated through somitogenesis, whereby epithelial somites bud off in pairs periodically from the rostral end of the unsegmented presomitic mesoderm (PSM). The periodicity of somitogenesis is governed by a molecular oscillator that drives periodic waves of clock gene expression caudo-rostrally through the PSM with a periodicity that matches somite formation. To date the clock genes comprise components of the Notch, Wnt, and FGF pathways. The literature contains controversial reports as to the absolute role(s) of Notch signalling during the process of somite formation. Recent data in the zebrafish have suggested that the only role of Notch signalling is to synchronise clock gene oscillations across the PSM and that somite formation can continue in the absence of Notch activity. However, it is not clear in the mouse if an FGF/Wnt-based oscillator is sufficient to generate segmented structures, such as the somites, in the absence of all Notch activity. We have investigated the requirement for Notch signalling in the mouse somitogenesis clock by analysing embryos carrying a mutation in different components of the Notch pathway, such as Lunatic fringe (Lfng), Hes7, Rbpj, and presenilin1/presenilin2 (Psen1/Psen2), and by pharmacological blocking of the Notch pathway. In contrast to the fish studies, we show that mouse embryos lacking all Notch activity do not show oscillatory activity, as evidenced by the absence of waves of clock gene expression across the PSM, and they do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for the formation of a segmented body axis.     

Monomeric APOBEC3G is catalytically active and has antiviral activity

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.     

Characterization of Nocardithiocin Derivatives Produced by Amino Acid Substitution of Precursor Peptide notG

International Journal of Peptide Research and Therapeutics - Nocardithiocin is a thiopeptide compound produced by the pathogenic actinomycete Nocardia pseudobrasiliensis that displays activity...     

Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli

Amino acid substitutions were made in the heat-labile enterotoxin signal sequence of Escherichia coli by recombinant DNA techniques, and their influence on the secretion of recombinant human epidermal growth factor by E. coli was examined. The heat-labile enterotoxin signal sequence is an amino-terminal extension of the octadecapeptide chain and is comprised of three distinct regions: a positively charged amino-terminal region, a central hydrophobic region, and a carboxyl-terminal region with the cleavage site recognized by the signal peptidase. Some alterations in the signal sequence caused a 1.5-3.5-fold increase in the secretion of recombinant human epidermal growth factor. These were the introduction of: (i) polar and small residues into the carboxyl-terminal region (replacement of Pro-1 Leu-3 with Asn-Ala or Ser-Ala), which may give a favorable structure for the recognition and cleavage by the signal peptidase; and (ii) a polar residue into the central hydrophobic region (replacement of Ile-9 with Ser), which may cause an increase of the affinity to the cytoplasmic membrane. In the latter case, a large amount of the unprocessed "precursor" was accumulated. The combination of these modifications, however, did not work additively. An increase in the amino-terminal positive charge (insertion of Lys) had no effect on secretion. These results prove that the level of protein secretion is greatly dependent on the polarity of the carboxyl-terminal region and the hydrophobicity and/or the amphiphilicity of the central region. Moreover, the overall balance of the physicochemical properties of respective regions is important.     

Synthesis of carbocyclic purine nucleosides using key intermediates, carbocyclic AICA-ribosides

Treating carbocyclic N1-methoxymethy-inosine and 2'-deoxy-inosine with 1N-NaOH/aq.EtOH gave the carbocyclic 5-amino-4-imidazolecarboxamide-riboside and -2'-deoxyriboside respectively. Reaction of both useful key intermediates with PhCONCS afforded the corresponding 5-(N-isothiocarbamoyl) derivatives in high yield. Methylation of the isothiocarbamoyl groups, followed by treatment with NaOH led to the purine ring-closure (guanine, isoguanine, and xanthosine) reaction.     

Effect of lung resection on blood lactate threshold in lung cancer patients

We studied the effect of a decrease in vital capacity (VC) on the blood lactate threshold detected during exercise in 16 preoperative (PRE) and 10 postoperative (POST) lung cancer patients who had undergone lobectomy or pneumonectomy. The PRE patients were selected on the basis of having normal preoperative pulmonary function. The POST patients were selected on the basis of having normal preoperative pulmonary function and a postoperative VC of less than 80%. The oxygen consumption/body surface area at a 2.2 m.mol.l-1 arterial lactate concentration (VO2/BSA at La-2.2) was adopted as the blood lactate threshold. VC/BSA in the POST group significantly correlated with VO2/BSA at La-2.2 (r = 0.85, P less than 0.01), but not in the PRE group. SaO2 at La-2.2 was 95.4 +/- 1.5% in the PRE group and 95.2 +/- 1.3% in the POST group. SaO2 at La-2.2 did not correlated with VC/BSA in either group. The hemoglobin concentration (Hb) in the arterial blood correlated significantly with VC/BSA in the POST group (r = 0.65, P less than 0.05) but not in the PRE group. These results indicate that VO2/BSA at La-2.2 was restricted by VC in patients with restrictive pulmonary function disorder. Of the three elements of oxygen delivery, Hb was a limiting factor for VO2/BSA at La-2.2 but SaO2 was not. Cardiac output, which was not measured in our study, was speculated to be another limiting factor for VO2/BSA at La-2.2.     

Photochromogenesis

The effect of light on the pigmentation of various strains of Nocardia, Corynebacterium, Arthrobacter, Brevibacterium, and Flavobacterium was investigated. It was revealed that thirty out of fifty-seven strains of Nocardia, two out of fifteen strains of Corynebacterium, two strains of Arthrobacter, six out of thirteen strains of Brevibacterium and two out of fourteen strains of Flavobacterium were photochromogenic; i. e., these strains produced yellow or orange pigments when grown under illumination but entirely unpigmented in total darkness. From these results, it may be concluded that photochromogenicity is not a particular phenomenon limited to specific species, but a common, widely distributed phenomenon in nonphotosynthetic bacteria.