The adipocyte-derived hormone, leptin, and its receptor, are now known to be integral components of a physiological signalling system that regulates fuel stores and energy balance. Constitutive leptin expression has been demonstrated only in adipose tissue, placenta and stomach. We have used RT-PCR to show that leptin mRNA is selectively transcribed in specific areas of rat brain and pituitary, and in a rat glioblastoma cell line. Using immunocytochemistry we have also shown leptin protein immunoreactivity in the corresponding tissues and cells, and confirmed this by Western blot using two epitope-specific antisera. Leptin mRNA expression in the hypothalamus is suppressed by fasting (48hr), suggesting a role for brain leptin in the central regulation of appetite. These data support the hypothesis that central nervous system derived leptin is a likely ligand for central leptin receptors.

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Revision of XGE-2019-2727R2" as invited by the action editor, Tim Curran More frequent, shorter trials enhance acquisition in a training session:There is a free lunch!

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A family of 16 genes encoding the mouse ribosomal protein S24 was identified, and four members from this family were cloned. A single expressed intron-containing S24 gene (termed mrpS24) and one pseudogene (mrpS24p) were completely sequenced and characterized. The mrpS24 gene has seven exons and six introns spanning over 5.1 x 10(3) nucleotides (nt). The cap site of S24 was mapped to a G residue four nt upstream of a polypyrimidine tract and 15 nt downstream of a TATA-like (TATGA) element. The 5' region (-325 to +33) of the mrpS24 gene has a functional promoter that was able to express the fused chloramphenicol acetyltransferase (CAT) reporter gene. Two different forms of mouse S24 cDNA clones were previously isolated. Sequence analysis showed that one of these cDNA clones (termed S24a) lacks the entire exon V sequence (18 nt), and the deduced amino acid sequence is missing a C-terminal lysine residue encoded by the other cDNA (S24b). The pseudogene mrpS24p is flanked by an 11-bp direct repeat, and its sequence is almost identical to the S24 cDNA sequence, but it lacks two mini-exons, V and VI (20 nt), as in the cases of the human and rat S24 cDNAs. RT-PCR experiments demonstrated the existence of a third form (S24c) that similarly lacks both of the mini-exons, and suggested that different species of S24 mRNA might arise from alternative splicing of the mini-exons V and VI. Northern blot analysis showed that S24 expression is down- and up-regulated during adipocyte differentiation and in cellular transformation, respectively. RNase protection assays and RT-PCR experiments suggested that these cell-specific changes of S24 mRNA levels are mainly due to fluctuations in S24c mRNA level. Our results provide the first indication that a ribosomal protein gene is regulated by alternative usage of two mini-exons in a cell-specific manner.

Vasoactive Intestinal Peptide (VIP) is the major physiological agonist of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride channel activity. VIP functions as a neuromodulator and neurotransmitter secreted by neurons innervating all exocrine glands. VIP is also a potent vasodilator and bronchodilator that regulates exocrine gland secretions, contributing to local innate defense by stimulating the movement of water and chloride transport across intestinal and tracheobronchial epithelia. Previous human studies have shown that the rich intrinsic neuronal networks for VIP secretion around exocrine glands could be lost in tissues from patients with cystic fibrosis. Our research has since confirmed, in vitro and in vivo , the need for chronic VIP exposure to maintain functional CFTR chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissues morphology [1] . The goal of the present study was to examine changes in VIP in the lung, duodenum and sweat glands of 8-and 17-weeks old F508del/F508del mice and to investigate VIPergic innervation in the small intestine of CF mice, before important signs of the disease development. Our data show that a low amount of VIP is found in CF tissues prior to tissue damage. Moreover, we found a specific reduction in VIPergic and cholinergic innervation of the small intestine. The general innervation of the primary and secondary myenteric plexus was lost in CF tissues, with the presence of enlarged ganglionic cells in the tertiary layer. We propose that low amount of VIP in CF tissues is due to a reduction in VIPergic and cholinergic innervation and represents an early defect that constitutes an aggravating factor for CF disease progression.

The CFTR chloride channel is regulated by phosphorylation at PKA and PKC consensus sites within its regulatory region (R-region) through a mechanism, which is still not completely understood. We used a split-CFTR construct expressing the N-term-TMD1-NBD1 (Front Half; FH), TMD2-NBD2-C-Term (Back Half; BH), and the R-region as separate polypeptides (Split-R) in BHK cells, to investigate in situ how different phosphorylation conditions affect the R-region interactions with other parts of the protein. In proximity ligation assays, we studied the formation of complexes between the R-region and each half of the Split-CFTR. We found that at basal conditions, the density of complexes formed between the R-region and both halves of the split channel were equal. PKC stimulation alone had no effect, whereas PKA stimulation induced the formation of more complexes between the R-region and both halves compared to basal conditions. Moreover, PKC + PKA stimulation further enhanced the formation of FH-R complexes by 40% from PKA level. In cells expressing the Split-R with the two inhibitory PKC sites on the R-region inactivated (SR-S641A/T682A), density of FH-R complexes was much higher than in Split-R WT expressing cells after PKC or PKC + PKA stimulation. No differences were observed for BH-R complexes measured at all phosphorylation conditions. Since full-length CFTR channels display large functional responses to PKC + PKA in WT and S641A/T682A mutant, we conclude that FH-R interactions are important for CFTR function. Inactivation of consensus PKC site serine 686 (S686A) significantly reduced the basal BH-R interaction and prevented the PKC enhancing effect on CFTR function and FH-R interaction. The phospho-mimetic mutation (S686D) restored basal BH-R interaction and the PKC enhancing effect on CFTR function with enhanced FH-R interaction. As the channel function is mainly stimulated by PKA phosphorylation of the R-region, and this response is known to be enhanced 34 |

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