Relationship between cell density and prolidase activity in human skin fibroblasts: effects of ascorbate and fructose

To evaluate the influence of cell density on the activity of fibroblast prolidase (EC 3.4.13.9), we determined this activity in sparse and dense cultures. We also investigated, the effects of different concentrations of β-d(?) fructose and l(+) ascorbate, which both increased cell density at confluency. For a fructose concentration of 25 mM, we observed that in the absence of glucose, intracellular total proteins increased 1.5-fold and prolidase specific activity, 1.8-fold. For ascorbate, a broad optimum concentration was found (range 0.01 – 0.50 mM). Addition to cultures of 0.1 mM ascorbate increased total proteins 1.4-fold, and doubled prolidase activity. This investigation was prompted by our previous results [J. Metab. Dis. 1983, 6, 27–31], confirmed here, and suggesting that increased prolidase activity at confluency was due to a rise in cell density.     

Apport de l’injection intracaverneuse de Prostaglandine E1 lors de la premiere consultation de l’impuissant: à propos de 414 cas

The authors report their experience of 414 injections of prostaglandin E1 (prostine). The diagnostic orientation between functional or organic impotence was assessed through questionaires and more particularly on the presence or absence of nocturnal or morning erections. The prostine reconditioning and self injection use techniques are precised: 80% of positive responses with better results in the oldest patients, whatever their medical antecedents. No relation between positive response to prostaglandin E1 and normal nocturnal erections could be found. Patients still presenting erections can present good or bad responses. This confirms the results previously given only allow us to put in evidence the good functional and anatomic state of cavernous bodies, without any etiological orientation (not mentioning the cases in which these self-injections reveal the presence of deformation, bend or fibrosis). The acceptance of self injection is low (30%) as well as a regular and prolonged medical observance (only 15%). This work confirms the low rate of local complications after repeated injections and the low rate of prolonged erections or priapism during Test 1. However, these possibilities must incite to prudence as for the generalization of this kind of treatment, out of specialized centers.     

Antipeptide antibody to the insulin-like growth factor-I receptor sequence 1232-1246 inhibits the receptor kinase activity.

To approach the question of why insulin-like growth factor-I (IGF-I) and insulin have different physiological actions, we developed antibodies directed against cytoplasmic regions of the IGF-I receptor exhibiting a low degree of homology with the corresponding sequences of the insulin receptor. We found that an antipeptide antibody directed against the beta-subunit carboxyl-terminal sequence (1232-1246) of the IGF-I receptor significantly reduced the in vitro receptor autophosphorylation. The ability of the synthetic peptide corresponding to the IGF-I receptor sequence 1232-1246 to abolish this inhibitory effect reflects the specific nature of the antibody interaction with the targeted domain in the receptor. Antipeptide antibody to IGF-I receptor sequence 1232-1246 also decreased receptor phosphorylation activity toward the exogenous substrate poly(Glu/Tyr). The reduction in poly(Glu/Tyr) phosphorylation was seen even when the antibody was incubated with a receptor previously activated and phosphorylated. Therefore, the inhibitory action on substrate phosphorylation is likely to be unrelated to the antibody reduction of receptor autophosphorylation but rather results from a global decrease in receptor enzymatic activity. The effect of the antipeptide antibody on receptor tyrosine kinase cannot be accounted for by a lowering of the receptor Km for ATP or of its affinity for the substrate poly(Glu/Tyr). Moreover, the interaction of the antibody with the receptor had no repercussion on the ligand binding site as shown by the unaltered IGF-I binding. Taken together our data suggest that the beta-subunit carboxyl-terminal domain of the IGF-I receptor plays a key role in regulating its kinase activity and that the particular sequence recognized by our antipeptide antibody could be involved in negative regulation of receptor functioning.     

The insulin receptor activation process involves localized conformational changes.

The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation.     

Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues

MicroRNAs (miRNAs) present in tissues and biofluids are emerging as sensitive and specific safety biomarkers. MiRNAs have not been thoroughly described in M. fascicularis, an animal model used in pharmaceutical industry especially in drug safety evaluation. Here we investigated the miRNAs in M. fascicularis. For Macaca mulatta, a closely related species of M. fascicularis, 619 stem-loop precursor miRNAs (pre-miRNAs) and 914 mature miRNAs are available in miRBase version 21. Using M. mulatta miRNAs as a reference list and homology search tools, we identified 604 pre-miRNAs and 913 mature miRNAs in the genome of M. fascicularis. In order to validate the miRNAs identified by homology search we attempted to sequence miRNAs expressed in kidney cortex from M. fascicularis. MiRNAs expressed in kidney cortex may indeed be released in urine upon kidney cortex damage and be potentially used to monitor drug induced kidney injury. Hence small RNA sequencing libraries were prepared using kidney cortex tissues obtained from three naive M. fascicularis and sequenced. Analysis of sequencing data indicated that 432 out of 913 mature miRNAs were expressed in kidney cortex tissues. Assigning these 432 miRNAs to pre-miRNAs revealed that 273 were expressed from both the -5p and -3p arms of 150 pre-miRNAs and 159 miRNAs expressed from either the -5p or -3p arm of 176 pre-miRNAs. Mapping sequencing reads to pre-miRNAs also facilitated the detection of twenty-two new miRNAs. To substantiate miRNAs identified by small RNA sequencing, 313 miRNAs were examined by RT-qPCR. Expression of 262 miRNAs in kidney cortex tissues ware confirmed by TaqMan microRNA RT-qPCR assays. Analysis of kidney cortex miRNA targeted genes suggested that they play important role in kidney development and function. Data presented in this study may serve as a valuable resource to assess the renal safety biomarker potential of miRNAs in Cynomolgus monkeys.