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Background: Since the discovery of autonomous leptin production in salivary glands, very few studies have reported on the physiological or pathological meaning of this particular cytokine in saliva. The aim of this study was to investigate the expression of leptin and its receptors Ob-Ra and Ob-Rb in parotid salivary gland tumors, with particular regard to a potential use of leptin as a tumor marker. Methods: Parotid tissue samples from healthy individuals (n = 31) and tumor patients (n = 97, including tissue samples from pleomorphic adenomas, adenolymphomas, basal cell adenomas, and diverse carcinomas) were analyzed by use of ApoTome-technique microscopy, immunohistochemistry, immunofluorescence, immunoblotting, and quantitative real-time PCR. Salivary and plasma leptin concentrations were measured by using ELISA. Ultrasound was used to determine tumor size before surgery. Results: In all salivary gland tumors leptin was expressed in much higher amounts than in healthy parotid tissues. The cytokine was not imported from the blood but actively produced by the tumors. Immunoblotting results indicated that leptin was present as oligomers in salivary glands. Furthermore, the examined tumors overexpressed the receptor isoforms Ob-Ra and Ob-Rb. Measured leptin concentrations in mixed saliva samples were significantly increased in patients with parotid tumors [mean (SD) 673 (484) pg/mL in pleomorphic adenomas, 679 (465) pg/mL in adenolymphomas, and 880 (618) pg/mL in carcinomas] vs controls [125 (36) pg/mL] (P < 0.001). Conclusions: This is the first study to show that the analysis of salivary leptin in mixed saliva samples may allow preoperative differentiation between tumor patients and healthy individuals.

The three different simple repetitive oligonucleotide probes (CT)8, (CAC)5 and (TCC)5 were hybridized to a panel of human DNAs which had been digested with the restriction endonucleases Alu I, Hinf I and Mbo I. The resulting DNA fingerprints were analyzed and different parameters calculated, such as the maximal mean allele frequency and the average number of polymorphic bands per individual. The highest number of bands was obtained after hybridization of Hinf I digested DNA with (CAC)5. The probability of finding the same band pattern as in individual A in individual B is 2 x 10(-8). The DNAs of monozygous twins show indistinguishable banding patterns and the bands are inherited according to the Mendelian laws. Thus this procedure reveals informative fingerprints that can be used for individual identification, e.g. in paternity testing and in forensic applications. In most of these experiments 32P-labelled probes were employed, yet the biotinylated oligonucleotide (GACA)4 produced results which were equivalent to those obtained by hybridization with the 32P-labelled probe (GACA)4.

A fast, reproducible and non-hazardous technique for non-isotopic DNA fingerprinting is presented. The method is based on digoxigenated oligonucleotides, which are specific for simple repetitive DNA sequences. The use of digoxigenin/anti-digoxigenin detection avoids many drawbacks inherent in e.g. the biotin/streptavidin system which often causes a poor signal-to-background ratio. Synthesis and purification of digoxigenated oligonucleotides and their use in filter hybridization are described in detail. Hybridization patterns obtained with four different radioactively labeled oligonucleotides have been compared with those of the respective digoxigenated probes. When slightly less stringent hybridization conditions are applied for digoxigenated oligonucleotides than for those labeled with 32P, the signal intensities are satisfying but additional minor bands occur as a result of the reduced stringency. With one explainable exception, these bands increase the information content of the fingerprint. In addition, hybridization of the digoxigenated (CAC)5 probe has been performed in situ with human metaphase chromosomes. The hybridization patterns in many mitoses resemble R-bands.

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Sixty eight individual clones of a human Y chromosome cosmid library were screened for the presence of GATA repeats, the major component of Bkm-related DNA sequences. Nine cosmid clones were found to cross-hybridize. The sequence organization of the repetitive base quadruplet GATA was analyzed using synthetic oligonucleotide probes. Subclones of GATA-positive cosmid clones were used for chromosomal localization of the Y-derived DNA sequences thus revealing male-specificity or male-female homology.

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