Biomedical subjects

Ferdinand S Vilim

Publications and source records attributed to Ferdinand S Vilim.

6 recordsLinked to original sources

PubMed2006

Identification of transcriptional regulators of neuropeptide FF gene expression.

Neuropeptide FF (NPFF) is an RF-amide peptide with pleiotropic functions in the mammalian central nervous system, including pain modulation, opiate interactions, cardiovascular regulation and neuroendocrine effects. To gain insights into the transcriptional mechanisms that regulate NPFF gene expression, we cloned and sequenced 9.8 and 1.5 kb of the mouse and rat NPFF 5'-flanking region, respectively. Regions with high sequence homology between mouse, rat and human were expected to have high probability to interact with regulatory proteins and were studied further. Electromobility shift assays revealed one region that may interact with the homeobox proteins Oct-1, PDX1, Pit-1 and MEIS and two consensus DRE sites that bind a nuclear protein, which was identified as the downstream regulatory element antagonistic modulator DREAM by supershift assays. The distribution of NPFF gene expression was examined in the mouse using in situ hybridization and RT-PCR. NPFF expression was also evident during mouse embryogenesis. A fixed transcription initiation site for the mouse NPFF gene was found. A novel splice variant with a retained intron of the NPFF gene was characterized. Chimeric luciferase reporter gene constructs for the mouse NPFF gene revealed a minimal promoter region and a region with transcriptional suppressor features. An NGF responsive area was found using mouse NPFF reporter gene constructs. We postulate that Oct-1, PDX1, Pit-1, MEIS and DREAM are likely transcriptional regulators of NPFF gene expression.

5' Flanking Region↗

PubMed2005

Identification of a new neuropeptide precursor reveals a novel source of extrinsic modulation in the feeding system of Aplysia.

The Aplysia feeding system is advantageous for investigating the role of neuropeptides in behavioral plasticity. One family of Aplysia neuropeptides is the myomodulins (MMs), originally purified from one of the feeding muscles, the accessory radula closer (ARC). However, two MMs, MMc and MMe, are not encoded on the only known MM gene. Here, we identify MM gene 2 (MMG2), which encodes MMc and MMe and four new neuropeptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to verify that these novel MMG2-derived peptides (MMG2-DPs), as well as MMc and MMe, are synthesized from the precursor. Using antibodies against the MMG2-DPs, we demonstrate that neuronal processes that stain for MMG2-DPs are found in the buccal ganglion, which contains the feeding network, and in the buccal musculature including the ARC muscle. Surprisingly, however, no immunostaining is observed in buccal neurons including the ARC motoneurons. In situ hybridization reveals only few MMG2-expressing neurons that are mostly located in the pedal ganglion. Using immunohistochemical and electrophysiological techniques, we demonstrate that some of these pedal neurons project to the buccal ganglion and are the likely source of the MMG2-DP innervation of the feeding network and musculature. We show that the MMG2-DPs are bioactive both centrally and peripherally: they bias egestive feeding programs toward ingestive ones, and they modulate ARC muscle contractions. The multiple actions of the MMG2-DPs suggest that these peptides play a broad role in behavioral plasticity and that the pedal-buccal projection neurons that express them are a novel source of extrinsic modulation of the feeding system of Aplysia.

Amino Acid Sequence↗

PubMed2003

Concerted GABAergic actions of Aplysia feeding interneurons in motor program specification.

GABAergic inhibitory interneurons regulate the activity of diverse types of neural networks, but the specific roles of these interneurons in motor control are poorly understood. In the Aplysia feeding motor network, three interneurons, cerebral-buccal interneuron-3 (CBI-3) and buccal interneurons B40 and B34, are GABA-immunoreactive and evoke fast IPSPs in their postsynaptic followers. Using a combination of pharmacological experiments with GABA antagonists, agonists, and uptake inhibitors, we found that these fast IPSPs are likely mediated by GABA. Functionally, these fast IPSPs specify two parameters for ingestive motor programs elicited by the command-like interneuron CBI-2: (1) the appropriate phasing of activity of the radula closer motor neuron B8 relative to protraction-retraction, and (2) protraction duration. First, in ingestive programs, B8 activity is phased such that it fires minimally during protraction. CBI-3 and B40 exert fast inhibition to minimize B8 activity during protraction, by either acting directly on B8 (B40) or indirectly on B8 (CBI-3). Second, these ingestive programs are characterized by long protraction duration, which is promoted by B40 and B34 because hyperpolarization of either cell shortens protraction. Such effects of B40 and B34 are attributable, at least partly, to their inhibitory effects on the retraction-phase interneuron B64 whose activation terminates protraction. Consistent with a GABAergic contribution to both B8 phasing and protraction duration, blockade of GABAergic inhibition by picrotoxin increases B8 activity during protraction and shortens protraction, without disrupting the integrity of motor programs. Thus, the concerted actions of GABAergic inhibition from three Aplysia feeding interneurons contribute to the specification of multiple features that define the motor program as an ingestive one.

Animals↗

PubMed2002

Analysis of human neuropeptide FF gene expression.

As an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7-kb sequence from the promoter region. Primer extension and 5'-rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U-251 MG, SK-N-SH, SK-N-AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real-time quantitative RT-PCR. The highest promoter activity was measured when a region from - 552 to - 830 bp of the 5'-flanking region was fused to the constructs, and a potential silencer element was localized between nucleotides -220 and -551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between - 61 and - 214 bp of the 5'-flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites.

3' Untranslated Regions↗

PubMed2002

Interneuronal and peptidergic control of motor pattern switching in Aplysia.

It has been proposed that a choice of specific behaviors can be mediated either by activation of behavior-specific higher order neurons or by distinct combinations of such neurons in different behaviors. We examined the role that two higher order neurons, CBI-2 and CBI-3, play in the selection of motor programs that correspond to ingestion and egestion, two stimulus-dependent behaviors that are generated by a single central pattern generator (CPG) of Aplysia. We found that CBI-2 could evoke either ingestive, egestive, or ambiguous motor programs depending on the regime of stimulation. When CBI-2 recruited CBI-3 firing via electrical coupling, the motor program tended to be ingestive. In the absence of CBI-3 activation, the program was usually egestive. When CBI-2 was stimulated to produce ingestive programs, hyperpolarization of CBI-3 converted the programs to egestive or ambiguous. When CBI-2 was stimulated to produce egestive or ambiguous programs, co-stimulation of CBI-3 converted them into ingestive. These findings are consistent with the idea that combinatorial commands are responsible for the choice of specific behaviors. Additional support for this view comes from the observations that appropriate stimulus conditions exist both for activation of CBI-2 together with CBI-3, and for activation of CBI-2 without a concomitant activation of CBI-3. The ability of CBI-3 to convert egestive and ambiguous programs into ingestive ones was mimicked by application of APGWamide, a neuropeptide that we have detected in CBI-3 by immunostaining. Thus combinatorial actions of higher order neurons that underlie pattern selection may involve the use of modulators released by specific higher order neurons.

Animals↗

PubMedDate not supplied

From precursor to final peptides: a statistical sequence-based approach to predicting prohormone processing.

Predicting the final neuropeptide products from neuropeptides genes has been problematic because of the large number of enzymes responsible for their processing. The basic processing of 22 Aplysia californica prohormones representing 750 cleavage sites have been analyzed and statistically modeled using binary logistic regression analyses. Two models are presented that predict cleavage probabilities at basic residues based on prohormone sequence. The complex model has a correct classification rate of 97%, a sensitivity of 97%, and a specificity of 96% when tested on the Aplysia dataset.

Amino Acid Sequence↗