The manuscript describes the “digital transcriptome atlas” of the developing mouse embryo, a powerful resource to determine co-expression of genes, to identify cell populations and lineages and to identify functional associations between genes relevant to development and disease.

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Analysis of the structure of nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex, site-directed mutagenesis studies and results from chemical modification of amino acid residues have identified loop 1, loop 4, and the N-terminal region of the NGF molecule as the most relevant for its biological activity. We synthesized several peptides mimicking the two loops (1 and 4) linked together with an appropriate spacer, with or without the N-terminal region. Two peptides named NL1L4 and L1L4 demonstrated good NGF agonist activity at a concentration as low as 3 M. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. In addition L1L4 was able to induce differentiation of PC12 cells. More interestingly, the peptide with the highest "in vitro" activity (L1L4) was shown to reduce neuropathic behavior and restore neuronal function in a rat model of peripheral neuropathic pain, thereby suggesting a potential therapeutic role for this NGF-mimetic peptide.

Angiopoietin-like (ANGPTL) proteins are secreted proteins showing structural similarity to members of the angiopoietin family. Some ANGPTL proteins possess pleiotropic activities, being involved in cancer lipid, glucose energy metabolisms, and angiogenesis. ANGPTL7 is the less characterized member of the family whose functional role is only marginally known. In this study, we provide experimental evidences that ANGPTL7 is over-expressed in different human cancers. To understand the role played by ANGPTL7 in tumor biology, we asked whether ANGPTL7 is endogenously expressed by malignant cells or in response to environmental stimuli. We found that ANGPTL7 is marginally expressed under standard growth condition while it is specifically up-regulated by hypoxia. Interestingly, the protein is secreted and partially associated with the exosomal fraction, suggesting that it could be found in the systemic circulation of oncologic patients and act in an endocrine way. Moreover, we found that ANGPTL7 exerts a pro-angiogenetic effect on human differentiated endothelial cells by stimulating their proliferation, motility, invasiveness, and capability to form capillary-like networks while it does not stimulate progenitor endothelial cells. Finally, we showed that ANGPTL7 promotes vascularization in vivo in the mouse Matrigel sponge assay, thereby accrediting this molecule as a pro-angiogenic factor.

We studied the effects of carbon starvation and of varying the growth rate on the activity of each of the two tandem ribosomal RNA promoters from the rrnA operon of Escherichia coli. The cellular abundance of plasmid-encoded transcripts arising at promoters P1 and P2 and terminating at the ribosomal RNA terminator in promoter-terminator fusions, together with transcript turnover rates, was used to estimate promoter activities. The rate of synthesis of the P1-promoted transcript was found to increase exponentially with growth rate and predominate at fast growth rates. The activity of the downstream promoter (P2) changed only slightly at different growth rates. Upon carbon starvation, little or no activity of the upstream promoter was detectable, while P2 activity persisted. We interpret this to mean that the dual promoters are differentially regulated so as to have separate adaptive and maintenance functions. This model simplifies most features of rRNA regulation known in E coli. In vitro studies have resulted in proposals of a variety of mechanisms by which rrn transcription regulation could occur (1, 2). In vivo studies have localized regulatory determinants with increasing precision. Fusions of rrn promoter regions with portions of the galactose operon have shown that the target for both stringent and growth rate control is within the promoter region (9, 10). Probably all of the E. coli rrn operons have dual promoters, although one remains that has not had its sequence determined. We have been able to measure the activity of each of the two rrinA promoters in vivo by using plasmids containing rrn promoter-terminator fusions (11,12). With rapidly growing cells, the upstream P1 promoter was found to be about three times more active than the downstream P2 promoter. During the stringent RNA control response, P1 was about 90% inhibited in relA+ but not in relA strains, indicating stringent control of P1 activity. In contrast, P2 was judged to be only moderately (50%) inhibited in both relA+ and relA hosts and, therefore, was not under stringent control. Furthermore, stringent regulation of P1 activity persisted even when the P2 promoter and the downstream regions extending to the mature 16S RNA gene were deleted (11,13).Here we describe measurements of the relative activities of the rrnA P1 and P2 promoters as a function of growth rate variation and during glucose starvation. We have found that the P1 promoter is strongly dependent on growth rate and can be progressively inactivated as growth slows to the point where, as during glucose starvation, it is only marginally detectable. The downstream P2 promoter behaves very differently; its activity is only weakly dependent on growth rate and remains quite active during glucose starvation. We suggest that the downstream promoter behaves as a constitutive maintenance promoter whose activity is relatively insensitive to regulation by either the stringent or growth-rate control mechanisms. This behavior can account for the excessive synthesis of rRNA in very slow...

Single-chain urokinase-type plasminogen activator or pro-urokinase is a zymogen with an intrinsic catalytic activity which is greater than that of most other zymogens. To study the structural basis for this activity, a three-dimensional homology model was calculated using the crystallographic structure of chymotrypsinogen, and the structure-function relationship was studied using site-directed mutagenesis and kinetic analysis. This model revealed a unique Lys300 in pro-urokinase which could form a weak interaction with Asp355, adjacent to the active site Ser356. It was postulated that this lysine, by its epsilon-amino group, may serve to pull Ser356 close to the active position, thereby inducing the higher intrinsic activity of pro-urokinase. This was consistent with the published finding that a homologous lysine (Lys416) in single chain tissue plasminogen activator when mutated to serine induced some reduction in activity. To test this hypothesis, a site-directed mutant with a neutral residue (Lys300-->Ala) was produced and characterized. The Ala300-pro-urokinase had a 40-fold lower amidolytic activity than that of pro-urokinase. It was also stable in plasma at much higher concentrations than pro-urokinase, reflecting much attenuated plasminogen activation. Plasmin activatability was comparable to that of pro-urokinase, but the resultant two-chain derivative (Ala300-urokinase) had a lower enzymatic activity (approximately 33% that of urokinase) due to a reduction of kcat. Interestingly, the KM of two-chain Ala300-urokinase against plasminogen was 5.8-fold lower than that of urokinase, being similar to that of pro-urokinase which has a KM about 5-fold lower than urokinase. In conclusion, the hypothesis that Lys300 is a key structural determinant of the high intrinsic activity of pro-urokinase was confirmed by these studies. This residue also appears to be important for the full expression of the enzymatic activity of urokinase.

Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion-exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2), and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C-terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the Cterminal portion of the molecule which is replaced by aspartic acid.Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin-like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported.The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides.HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.Hirudin, the most potent thrombin inhibitor known, is a small polypeptide discovered over 100 years ago in the saliva of the medicinal leech Hirudo medicinalis [l]. It was first characterized biochemically by Markwardt [2, 31 and its structure was lately elucidated by Dodt et al. [4]. It is a single-chain polypeptide composed of 65 amino acids, including 6 cysteine residues, and a molecular mass of about 7000 Da. Hirudin produces its action by binding directly to thrombinCorrespondence to E. Scacheri,

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