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Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5' untranslated region (5'-UTR). A single 5'-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5'-UTR is mutually exclusive. The short and long 5'-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5'-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.

By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSLtes. Due to an addition of amino acids at the NH2-termini, rat and human HSLtes consist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSLadi). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSLadi. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSLadi sequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M(r) approximately 120,000) that exhibited catalytic activity similar to that of HSLadi. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells.

The existence of a DNA polymorphism at the hormone-sensitive lipase locus could be of great interest for genetic analysis of obesity and related disorders since hormone-sensitive lipase is the rate-limiting enzyme of adipose tissue lipolysis and therefore plays a key role in energy metabolism. The polymorphic dinucleotide repeat D19S120 was identified within a human genomic clone selected with a rat hormone-sensitive lipase cDNA. This marker was subsequently localized to the short arm of chromosome 19 (p13.3) whereas human hormone-sensitive lipase (LIPE) had been mapped to the long arm of chromosome 19 (q13.1-->13.2). A duplication of the hormone-sensitive lipase gene or the presence of a pseudogene could explain the discrepancy. Cosmids from the two regions were analyzed in Southern blot experiments. A human adipose tissue hormone-sensitive lipase full-length cDNA probe hybridized only to cosmids from the 19q13.1-->13.2 region whereas the D19S120 amplicon probe hybridized only to cosmids from the p13.3 region. These data show that the occurrence of gene duplication or the presence of a pseudogene on the short arm of chromosome 19 is very unlikely and that D19S120 is unrelated to the hormone-sensitive lipase gene.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.

The aim was to study hang-over effects from alcohol in simulated driving. The driving task was to negotiate 20 km in as short time as possible. The road had many curves, horizontal and vertical, with ice friction at irregular intervals. 24 healthy volunteers, aged 22-46, with valid driving licenses, all screened as moderate drinkers, participated as subjects in a repeated measurements design. In the acute intoxicated state (average BAC 150 mg%), driving performance was severely impaired. Performance was also impaired, but to a much lesser degree, in the morning after the alcohol consumption when average BAC was just below 40 mg%. Later during the day no significant differences were demonstrated. After another night's sleep, all effects were gone. An issue raised in a previous study on hang-over effects, whether the subjects were treated exactly alike in the hang-over and the control conditions the night before testing (with the exception for the alcohol consumption in the first case), that is attended a party and stayed overnight at the research institute in both conditions, or whether they did so only in the hang-over condition, did not seem to matter for the estimated hang-over effect.

Eighteen healthy volunteers of both sexes, aged 20-35 years, were tested in the morning after three nights of medication with brotizolam 0.25 mg, nitrazepam 5 mg or placebo on a monotonous simulated driving task. The effect measures were subsidiary auditory reaction time and time outside road. Measurements of self-rated alertness were carried out as well. No effects were demonstrated from treatments on either measure. Nitrazepam however tended to score worst on all measures, except time outside road which could not be analysed with respect to statistical significance because of an insufficient number of subjects leaving the road. Twelve of the subjects were also tested immediately after drug intake on the first night of each medication period. Reaction time decrement was observed in both active drugs conditions with no difference between the two. The other measures, however non-significant, pointed in the same direction with the greatest decrement for nitrazepam.

Drivers suffering from sleepiness at the wheel run the risk of being involved in car accidents. To evaluate whether objective data of driving performance can be assessed in patients with excessive tendency of falling asleep at the wheel, two test versions of a computerized driving program were created to fit an advanced driving simulator. For the evaluation 15 male drivers with habitual sleep spells whilst driving were selected among patients with the clinical features of the sleep apnoea syndrome. The brake reaction time and the deviations from straight road-line were significantly increased when compared to the performance of 10 matched controls. Irrespective of test version, the driving simulator with the program used was found to be a sensitive method to ascertain driving vigilance impairment in quantitative terms. It would also be a valuable method to evaluate the efficiency of treatment in selected patients.

Eighteen healthy volunteers of both sexes, aged 20-34, were tested in the morning while undertaking real car driving avoidance manoeuvres and during monotonous simulated driving after 1 and 3 nights of medication with triazolam 0.25 mg, nitrazepam 5 mg or placebo. The study was a double-blind, randomized, cross-over study, where a minimum of 7 days wash-out separated the 3 treatment periods. Nitrazepam was found to impair performance in the simulated task after 1 but not after 3 nights of medication. Performance in the triazolam condition was not significantly different from the other conditions on this task on either day. However, after one night of medication triazolam tended to score worse than placebo but better than nitrazepam. In real car driving a tendency was noted for nitrazepam to score worst, whereas the difference between placebo and triazolam was hardly noticeable. The same tendency appeared on both days.

The composition of the eosinophilic cell series in the bone marrow has been analysed in 10 patients with a pronounced reactive eosinophilia (RE) and in 2 with eosinophilic leukaemia (EL). An imparied differentiation of the eosinophils was found in the EL patients compared with the RE group. Thus the ratio of eosinophilic promyelocytestmyelocytes: segmented eosinophils was 9.2 and 9.1, respectively, in the patients with EL and 0.1--3.1 (average 1.3) in the RE patients. It is suggested that EL is characterized by an impaired differentiation of the eosinophilic bone marrow cells and that the recognition of this abnormality is of value in the diffential diagnosis between EL and RE.