Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats.

The obese (ob) gene has recently been isolated through a positional cloning approach, the mutation of which causes a marked hereditary obesity and diabetes mellitus in mice. In the present study, we isolated rat ob cDNA and examined the tissue distribution of the ob gene expression in rats. We also studied the gene expression in genetically obese Zucker fatty (fa/fa) rats. The rat ob gene product, a 167 amino acid protein with a putative signal sequence, was 96 and 83% homologous to the mouse and human ob proteins, respectively. Northern blot analysis using the rat ob cDNA probe identified a single mRNA species of 4.5 kb in size in the adipose tissue, while no significant amount of ob mRNA was present in other tissues in rats. The ob gene was expressed in the adipose tissue with region specificities. The rank order of the ob mRNA level in the adipose tissue was epididymal, retroperitoneal, and pericardial white adipose tissue > mesenteric and subcutaneous white adipose tissue > or = interscapular brown adipose tissue. The ob gene expression occurred in mature adipocytes rather than in stromalvascular cells isolated from the rat adipose tissue. Expression of the ob gene was markedly augmented in all the adipose tissue examined in Zucker fatty (fa/fa) rats at the stage of established obesity. The present study leads to the better understanding of the physiologic and pathophysiologic roles of the ob gene.

Human obese gene expression. Adipocyte-specific expression and regional differences in the adipose tissue.

The obese (ob) gene, the mutation of which results in severe hereditary obesity and diabetes in mice, has recently been isolated through positional cloning. In this study, we isolated a full-length human ob complementary DNA (cDNA) clone and examined the tissue distribution of ob gene expression in humans. The nucleotide sequences of the human ob cDNA coding region were 83% identical to those of the mouse and rat ob cDNA coding regions. Analysis of the deduced amino acid sequences revealed that the human ob protein is a 166-amino acid polypeptide with a putative signal sequence and is 84 and 83% homologous to the mouse and rat ob proteins, respectively. Northern blot analysis using the cloned human ob cDNA fragment as a probe identified a single messenger RNA (mRNA) species 4.5 kb in size found abundantly in the adipose tissues obtained from the subcutaneous, omental, retroperitoneal, perilymphatic, and mesenteric fat pads. However, no significant amount of ob mRNA was present in the brain, heart, lung, liver, stomach, pancreas, spleen, small intestine, kidney, prostate, testis, colon, or skeletal muscle. The ob mRNA level in the adipose tissue varied from region to region even in the same individual. Furthermore, in the human adipose tissue, ob gene expression occurred in mature adipocytes rather than in stromal-vascular cells. This study is the first report of the elucidation of ob gene expression in human tissues, thereby leading to better understanding of the physiological and clinical implications of the ob gene.

Molecular screening of both the promoter and the protein coding regions in the human ob gene in Japanese obese subjects with non-insulin-dependent diabetes mellitus.

OBJECTIVE: Although the molecular mechanism of obesity has been poorly understood, recent studies indicate that leptin plays a critical role in regulating both food intake and body weight. Because obesity decreases the sensitivity to insulin, the human ob gene is presumed to be one of the candidate genes for non-insulin-dependent diabetes mellitus (NIDDM) associated with obesity. Although the protein coding region in the ob gene has been screened for mutations, the promoter region and the non-coding first exon have not yet been studied. We investigated the involvement of the human ob gene, especially mutations at the promoter region and the non-coding first exon, in the development of NIDDM associated with obesity. SUBJECTS: The study group comprised 60 Japanese obese subjects with NIDDM (body mass index (BMI) 43.6 > or = BMI > or = 26.4, 29.0+/-0.41 (mean+/-S.E.M.)) and 24 obese individuals with impaired glucose tolerance (IGT) (30 > or = BMI > or = 26.4, 27.1+/-0.22). METHODS: Mutations at both the promoter region and all three exons in the human ob gene were screened by the single-stranded conformational polymorphism analysis. When aberrantly migrated bands were recognized, the PCR-amplified DNA fragment was directly sequenced. RESULTS: In the protein coding region a silent mutation in the second exon was detected. The non-coding first exon and the about 100 bp 5'-flanking region of the gene which contains a proximal CCAAT/enhancer-binding protein site were screened, but no mutations were found. CONCLUSION: These results suggest that no mutations in either the promoter region at the about 100 bp 5'-flanking region of the gene, or in any of the three exons, are involved in the development of NIDDM or IGT associated with obesity.

Endoscopic forehead lift. Evolution and update.

This article reviews the author's experience with the endoscopic forehead lift. The basic technique, patient selection criteria, and advantages and disadvantages are presented.

Endoscopic facial rejuvenation.

The use of an endoscope is a reliable way to perform facial rejuvenation, to correct eyebrow imbalance, periorbital and malar soft-tissue sagginess, and soft-tissue displacement in the lower face and neck. It allows great magnification and tissue dissection control while observing in situ tissue modification and the immediate results. In mild cases of brow ptosis without muscle hyperactivity, wide undermining and fixation can be a simple way to achieve a good result. When hyperactivity of the muscles is present with or without brow ptosis, a more complex procedure can be done, which requires the treatment of the appropriate musculatures. The result are comparable to those achieved with the open approach with the advantage of a shorter scar, minimal permanent numbness, and much better patient acceptability.

The corrugator supercilii muscle revisited.

BACKGROUND: Few studies have examined the anatomy of the central forehead as it pertains to vertical glabellar furrows. OBJECTIVE: This study sought to critically examine the corrugator supercilii muscle (CSM) in situ, in relation to surrounding bony and soft tissue landmarks. METHODS: Anatomic dissection of 10 fresh cadaveric hemi-heads was performed, focusing on the CSM origin and insertion, with emphasis on regional fascial relations, neurovascular structures, and osseous topography. RESULTS: The CSM originates along the supraciliary arch. The muscle is attached at its medial and superior margin, whereas the lateral and inferior margins are free. The muscle originates from a bony plateau on the supraciliary arch. The CSM travels laterally, with most of the muscle passing through the fibers of the orbicularis oculi and the frontalis. The dermal insertion of the muscle is under the central portion of the eyebrow. The nerve supply enters at the lateral aspect of the muscle approximately 5 mm cephalic to the lateral brow. The action of the CSM is to elevate the medial aspect of the brow and depress the lateral segment of the brow. CONCLUSION: The CSM does not appear to be the primary determinant of vertical glabellar frown lines. (Aesthetic Surg J 2001;21:209-215.).

Endoscopic facial rejuvenation: endoforehead, the functional lift. Case reports.

Endoscopic techniques have only very recently been applied to aesthetic plastic surgery procedures, especially in carpal tunnel release, in forehead plasties, and in breast augmentation operations. The author briefly outlines his experiences with endoscopic forehead lifts, first reported at surgical conferences in Los Angeles and Buenos Aires in 1992, and now totaling 61 cases performed in the 11 months just prior to the submission of this article. This endoscopic approach to forehead lifting has provided similar and comparable results to conventional coronal forehead lift operations, by means of small incisions made in the scalp area with minimal and fewer complications.

Structural organization and chromosomal assignment of the human prostacyclin receptor gene.

Prostacyclin receptor is a member of the prostanoid receptor family in the G protein-coupled receptor superfamily with seven transmembrane domains. We report here the isolation and structural organization of the human prostacyclin receptor gene. Southern blot analysis demonstrated a single copy of the human prostacyclin receptor gene in the human genome. The human prostacyclin receptor gene spanned approximately 7.0 kb and was composed of three exons separated by two introns. The first intron occurred in the 5'-untranslated region, 13 bp upstream to the ATG start codon. The second intron was located at the end of the sixth transmembrane domain, thereby separating it from the downstream coding region and the 3'-untranslated region. By primer extension analysis, the transcription initiation sites were mapped 870-872 bp upstream to the ATG start codon. The 1.2-kb human prostacyclin receptor 5'-flanking region lacked conventional TATA and CCAAT boxes, but it contained several cis-acting regulatory elements including an inverted CCAAT box (Y box) and two copies of SP-1 binding sites. Using human-rodent somatic hybrid cell DNA, the human prostacyclin receptor gene was assigned to human chromosome 19. The present study helps establish the genetic basis for prostacyclin receptor research and provides further insight into the molecular mechanisms underlying the prostanoid receptor family.

Structural organization and chromosomal assignment of the human obese gene.

The obese (ob) gene has been identified through a positional cloning approach; the mutation of this gene causes marked hereditary obesity and diabetes mellitus in mice. We report here the isolation and characterization of the human ob gene. Southern blot analysis demonstrated a single copy of the ob gene in the human genome. The human ob gene spanned approximately 20 kilobases (kb) and contained three exons separated by two introns. The first intron, approximately 10.6 kb in size, occurred in the 5'-untranslated region, 29 base pair (bp) upstream of the ATG start codon. The second intron of 2.3 kb in size was located at glutamine +49. By rapid amplification of 5'-cDNA ends, the transcription initiation sites were mapped 54-57 bp upstream of the ATG start codon. The 172-bp 5'-flanking region of the human ob gene contained a TATA box-like sequence and several cis-acting regulatory elements (three copies of GC boxes, an AP-2-binding site, and a CCAAT/enhancer-binding protein-binding site). By the fluorescence in situ hybridization technique, the ob gene was assigned to human chromosome 7q31.3. This study should establish the genetic basis for ob gene research in humans, thereby leading to the better understanding of the molecular mechanisms underlying the ob gene.

Molecular cloning of rat leptin receptor isoform complementary DNAs--identification of a missense mutation in Zucker fatty (fa/fa) rats.

We cloned the full-length rat leptin receptor (Ob-R) isoform complementary DNAs (cDNAs) and examined the gene expression in rats. We also identified a mutation in Ob-R in Zucker fatty (fa/fa) rats. Three alternatively spliced isoforms (Ob-Ra, Ob-Rb, and Ob-Re) have been identified, which are closely related to the gp130 signal-transduction component of class I cytokine receptors. Rat Ob-Ra and Ob-Rb were single transmembrane proteins, which differ in the C-terminal amino acid sequences. On the other hand, Ob-Re had no transmembrane domain and was a soluble form of the receptor. Reverse transcription-polymerase chain reaction analysis revealed that Ob-R isoform messenger RNAs (mRNAs) are expressed in a wide variety of rat tissues in tissue-specific manners. A missense mutation (an A to C conversion at nucleotide position 806) was found in the extracellular domain of all the isoforms in Zucker fatty (fa/fa) rats, which resulted in an amino acid change from Gln to Pro at + 269 (the Gln269Pro mutation). These Ob-R isoform mRNAs were present in the brain from Zucker fatty (fa/fa) rats at comparable amounts to those in their lean littermates. The present study provides new insight into the molecular mechanisms for Ob-R.

Genomic organization, expression, and chromosomal mapping of the mouse adrenomedullin gene.

We have isolated and characterized the mouse adrenomedullin (AM) gene (Adm) and determined its chromosomal location. The gene spans approximately 2.1 kb and is organized into four exons separated by three introns. The transcription start site was determined to be the adenine nucleotide at -618. The mouse AM 5'-flanking region contains a TATA box-like sequence and several cis-acting regulatory elements. Analysis of the nucleotide and deduced amino acid sequences revealed that mouse preproAM is a 184-amino-acid polypeptide, from which AM and proAM N-terminal 20 peptide are cleaved. Using restriction fragment length variants on a DNA panel of interspecific backcross mice, we mapped Adm to a distal region of mouse chromosome 7.

Identification of the human leptin 5'-flanking sequences involved in the trophoblast-specific transcription.

Leptin is an adipocyte-derived blood-borne satiety factor that is involved in the regulation of energy homeostasis. We have recently demonstrated nonadipose tissue production of leptin; leptin is synthesized in and secreted from placental trophoblasts (Nature Med. 3: 1029-1033, 1997). To understand the transcriptional regulation of the human leptin gene in placental trophoblasts, we examined the promoter activity of various lengths of the human leptin 5'-flanking sequences in BeWo cells, a human trophoblastic cell line. The 2080-bp human leptin gene promoter region (-2080 to +108) showed a high-level transcription activity in BeWo cells. When DNA sequences between -1885 and -1830 were deleted, the promoter activity was reduced dramatically in BeWo cells. No significant changes in the promoter activity were noted when tested in primary cultures of rat mature adipocytes. Electrophoretic mobility shift assays revealed the presence of nuclear protein(s) binding to the sequences in BeWo cells but not in isolated rat mature adipocytes. The present study provides new insight into the trophoblast-specific transcription of the human leptin gene.