New and unexpected cosmetic properties of perfumes. Effects upon free radicals and enzymes induced by essential oils, absolutes and fragrant compounds.

The biological properties of several different perfume components have been investigated. It has been demonstrated, through appropriate test methods, that essential oils, absolutes and even compounds show significant (anti/pro)-radical, (anti/pro)-elastasic and (anti/pro)-tyrosinasic activities. These unexpected properties open up new opportunities for the formulation of cosmetic products and could contribute to the understanding of activities traditionally attributed to essential oils by Aromatherapy.

Coexpression of G-CSF with an unglycosylated G-CSF receptor mutant results in secretion of a stable complex.

Previously, we have shown that the entire extracellular domain of the granulocyte-colony stimulating factor receptor (sG-CSFr) produced in Chinese hamster ovary (CHO) cells forms a stable complex with its ligand G-CSF, at a stoichiometry of 2:2. A truncated receptor molecule consisting of the cytokine receptor homology domain and N-terminus Ig-like domain (Ig CRH) behaves quite similarly. Both of these forms of the receptor are highly glycosylated. To address the importance of glycosylation toward receptor activity and stability, and possibly obtain nonglycosylated receptor for crystallization, mutations were made to replace four Asn residues which are N-glycosylated in the truncated receptor. Virtually no receptor was recovered from conditioned media of CHO cells transfected with this mutant construct, although a high-level of mRNA coding for receptor was detected; this mRNA was translated as determined by Western blots of cell lysates. These results indicate that the translated product is apparently not secreted from these cells. Cells transfected with mutant receptor cDNA were cotransfected with a cDNA construct expressing G-CSF in which the single O-glycosylation site was eliminated by mutation. Upon fermentation of the cotransfectants, we observed a large amount of receptor-ligand complex in the conditioned media. The purified unglycosylated complex appeared to be of the same binding stoichiometry and approximate binding affinity as that of complex formed by addition of purified ligand and unmutated receptor. These results show that while glycosylation of sG-CSFr is not necessary for ligand binding, it appears to be crucial in folding and export from the cell.

Oxidative stress status and antioxidant status are apparently not related to carotenoid status in healthy subjects.

Several lines of evidence suggest that carotenoids may have a beneficial effect on health as a result of their antioxidant properties. In addition to beta-carotene, five other carotenoids are recovered in noticeable amounts from human plasma and tissues. Although the effect of beta-carotene on in vivo lipid peroxidation has been documented, few data are available on the effects of the other carotenoids. We evaluated the ability of the main human carotenoids to reduce lipid peroxidation by determining the correlations between plasma carotenoid concentration and plasma antioxidant capacity (in 79 healthy volunteers) and between carotenoid status and breath pentane excretion (in a subgroup of 24 subjects). Carotenoid intake was assessed by means of a 3-day food recall. Carotenoid status was evaluated by measurement of beta-carotene, lycopene, lutein/zeaxanthin, and alpha-carotene in plasma and buccal mucosal cells. Antioxidant status was evaluated by measurement of the total antioxidant capacity of the plasma. Oxidative stress status was evaluated by breath pentane measurements. Food recall data and the carotenoid concentrations in plasma and buccal mucosal cells showed that the subjects had normal carotenoid intake and normal carotenoid status. The total antioxidant capacity of the plasma was not related to the concentration of any specific carotenoid. The level of expired air pentane was not related to the carotenoid status of the subjects. These results show that normal concentrations of carotenoids in plasma and tissues are not correlated with these clinical markers of antioxidant and oxidative stress status.

Amyloid precursor protein processing in sterol regulatory element-binding protein site 2 protease-deficient Chinese hamster ovary cells.

Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.

Expression of nonmuscle myosin heavy chain-B isoform in the vessel wall of porcine coronary arteries after balloon angioplasty.

Nonmuscle myosin heavy chain-B isoform (NMMHC-B) is expressed by proliferating vascular smooth muscle cells (SMCs), and its expression in primary lesions has been proposed to be predictive of restenosis after atherectomy. The present study was designed to study the time-course expression of NMMHC-B after angioplasty of porcine coronary arteries by in situ hybridization and immunohistochemistry. Domestic juvenile swine underwent percutaneous transluminal coronary angioplasty (PTCA) of the left anterior descending and circumflex coronary arteries with standard clinical angioplasty catheters. To identify proliferating cells, 5'-bromo-2'-deoxyuridine (BrdU) was administered and detected by immunohistochemistry on serial sections. Vessels were examined at 3, 7, and 14 days after balloon angioplasty, and uninjured coronary vessels were used as controls. Normal arteries showed hybridization to 35S-labeled NMMHC-B riboprobes localized mainly in the medial layer. NMMHC-B expression in the adventitia was markedly increased 3 days after balloon angioplasty. Seven and 14 days after injury, NMMHC-B mRNA-containing cells were localized in the adventitia and neointima at the arterial injury site. Cell proliferation, as indicated by BrdU staining, colocalized with NMMHC-B mRNA expression 3 and 7 days after angioplasty. These data indicate that cells proliferating in the adventitia and neointima express NMMHC-B; however, its expression is not limited to the proliferative state, since NMMHC-B mRNA was also found in quiescent SMCs of normal coronary arteries and in nonproliferating adventitial and neointimal cells 14 days after angioplasty.

Dimerization of granulocyte-colony stimulating factor receptor: the Ig plus CRH construct of granulocyte-colony stimulating factor receptor forms a 2:2 complex with a ligand.

We have previously shown that the extracellular domain of granulocyte-colony stimulating factor receptor (soluble G-CSFR), prepared from CHO cell conditioned media, dimerizes upon binding its ligand, G-CSF. The most stable ligand-receptor complex occurs at a 2:2 stoichiometry, unlike the growth hormone and erythropoietin systems. In the latter cases, each ligand uses two sites to bring two receptors together. In this study, we have generated a truncated G-CSF receptor, known to be sufficient for high affinity ligand binding, which consists of an Ig-like domain and a cytokine receptor homology module. With an affinity purified receptor, sedimentation equilibrium experiments clearly demonstrated that this truncated form of the receptor behaves very similarly to the entire extracellular domain. The sedimentation equilibrium data are consistent with the model that the truncated receptor has a weak tendency to self-associate into a dimer in the absence of a ligand, this receptor-receptor interaction is enhanced by ligand binding, and the most stable complex occurs at a 2:2 stoichiometry. These results are very different from those described by others for various murine G-CSF receptor constructs from either Escherichia coli or insect expression systems.

Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries.

BACKGROUND: In the present series of experiments, we examined the onset of cell proliferation and growth factor expression after balloon overstretch injury to porcine coronary arteries. METHODS AND RESULTS: Domestic juvenile swine underwent balloon overstretch injury to the left anterior descending and circumflex coronary arteries with standard percutaneous transluminal coronary angioplasty balloon catheters. To identify proliferating cells, 5-bromo-2-deoxyuridine (BrDU) was administered over a period of 24 hours before the animals were killed at either 1, 3, 7, or 14 days after injury. Immunohistochemistry was performed with monoclonal antibodies to BrDU and smooth muscle cell markers. Three days after injury, a large number of proliferating cells were located in the adventitia, with significantly fewer positive cells found in the media and lumen. Seven days after injury, proliferating cells were found primarily in the neointima, extending along the luminal surface. In situ hybridization for PDGF A-chain and beta-receptor mRNAs revealed that the expression of these two genes was closely correlated with the sites of proliferation at each time point. Studies in which BrDU was injected between days 2 and 3 and the animals were killed on day 14 suggested that the proliferating adventitial cells may migrate into the neointima. CONCLUSIONS: These data suggest that adventitial myofibroblasts contribute to the process of vascular lesion formation by proliferating, synthesizing growth factors, and possibly migrating into the neointima. Increased synthesis of alpha-smooth muscle actin observed in the adventitial cells after arterial injury may constrict the injured vessel and contribute to the process of arterial remodeling and late lumen loss after angioplasty.

[Tuberculosis: multidrug-resistance in Belgium in 1992 and 1993]. / Tuberculose: la multirésistance en Belgique en 1992 et 1993.

The "Belgian TB Multidrug Resistance Working Group" determined in collaboration with 28 laboratories carrying out antibiograms for mycobacteria, the prevalence and incidence of multidrug resistance in Belgium in 1992-1993. During this period, respectively 14 (1.1%) and 17 (1.3%) cases of multidrug resistance (i.e. resistance to at least isoniazid and rifampicin, according to the W.H.O. definition), were detected by these laboratories. Since 9 new cases of multidrug resistance were detected in 1992 and 10 in 1993, the incidence of multidrug resistance in Belgium can be estimated at 0.1 per 100.000 inhabitants. Among these 19 new cases, 2 are confirmed as primary resistance cases.

Increase of singlet oxygen protection of erythrocytes by vitamin E, vitamin C, and beta carotene intakes.

Substantial evidence supports the theory that free radicals, especially oxygen radicals, are involved in the process of aging. The human organisms have two ways to fight them: an enzymatic way with enzymatic intervention like superoxide dismutase, catalase... and a chemical way with the intervention of scavengers such as vitamins, cysteine, methionine, gluthatione... The aim of this work was to determine that an intakes of vitamins association: vitamin E, vitamin C and beta carotene induce an increase of singlet oxygen protection of erythrocytes' subjects. The method was based on the haemolytic effect of singlet oxygen which is generated by irradiation of hematoporphyrine at 365 nm, in 22 p. cent suspension of erythrocytes' subjects. Results show that a supply of beta carotene (15 or 30 mg/day), vitamin E (15 mg/day) and vitamin C (30 mg/day) involves an increase of singlet oxygen protection of erythrocytes of subjects. This protection appears very quickly after 15 days of treatment.

Hypertensive strains and normotensive 'control' strains. How closely are they related?

The spontaneously hypertensive rat and the Dahl salt-sensitive rat are the most widely studied genetic models of hypertension. Many investigators have attempted to study the pathogenesis of hypertension by comparing these strains with their respective normotensive "controls," the Wistar-Kyoto rat and the Dahl salt-resistant rat. However, the genetic relation between each of these hypertensive strains and its corresponding normotensive control has never been clearly defined. Based on an analysis of DNA "fingerprint" patterns generated with six multilocus probes, we found that the spontaneously hypertensive rat (Charles River Laboratories, Inc.) is genetically quite different from its normotensive Wistar-Kyoto control: these strains only share approximately 50% of their DNA fingerprint bands in common. The inbred Dahl salt-sensitive rat (SS/Jr strain) (Harlan Sprague Dawley, Inc.) and the Dahl salt-resistant rat (SR/Jr strain) share approximately 80% of their DNA fingerprint bands in common. To the extent that the genes identified by DNA fingerprint analysis are representative of loci dispersed throughout the rodent genome, the current findings provide evidence of extensive genetic polymorphism between these commonly used hypertensive strains and their corresponding normotensive controls, particularly in the spontaneously hypertensive rat model. These findings, together with the fact that an enormous number of biochemical and physiological differences have been reported between these hypertensive and normotensive strains, suggest that continued comparison of spontaneously hypertensive rats with Wistar-Kyoto rats or Dahl salt-sensitive with salt-resistant rats will have limited value for investigating the pathogenesis of hypertension.

Assignment of rat linkage group V to chromosome 19 by single-strand conformation polymorphism analysis of somatic cell hybrids.

The rat provides a number of important models of human genetic disease; however, the rat genetic map has not been extensively developed. Although most rat chromosomes carry several gene assignments, some major linkage groups (LG) remain to be mapped. To determine the chromosome location of the largest unmapped linkage group in the rat (LG V containing multiple carboxylesterase loci), we used single-strand conformation polymorphism analysis to identify the rat esterase-10 gene in a panel of rat x mouse somatic cell hybrids. We found that the carboxylesterase gene family and hence LG V are located on rat chromosome 19. We have also confirmed the assignment of the angiotensinogen gene to rat chromosome 19 and have used a large set of recombinant inbred strains to map two anonymous variable number of tandem repeat (VNTR) markers to this chromosome. The current findings bring the total number of genes assigned to rat chromosome 19 from 3 to 19 and provide further evidence of substantial homology between this chromosome and chromosome 8 in the mouse.

Sequence analysis of the alpha 1 Na+,K(+)-ATPase gene in the Dahl salt-sensitive rat.

In the inbred Dahl salt-sensitive rat (SS/Jr strain), it has been proposed that a T for A transversion in the DNA sequence encoding amino acid 276 in the alpha 1 subunit isoform of Na+,K(+)-ATPase may impair ion transport and contribute to the pathogenesis of hypertension. This hypothesis is of major scientific interest because it represents the first attempt to explain the pathogenesis of salt-sensitive hypertension on the basis of a specifically defined mutation at the DNA level. We devised a polymerase chain reaction technique to screen the genomic DNA of multiple SS/Jr rats for the T for A transversion reported in the complementary DNA (cDNA) encoding the alpha 1 subunit of Na+,K(+)-ATPase. When eight Dahl SS/Jr rats from Harlan Sprague Dawley Inc. were tested with the polymerase chain reaction technique, we found no evidence of this mutation in the Na+,K(+)-ATPase gene. Direct sequence analysis of the gene in three SS/Jr rats also did not show the T for A transversion. These results 1) strongly suggest that commercially available Dahl SS/Jr rats do not carry a T for A transversion in the genomic DNA sequence encoding amino acid 276 in the alpha 1 subunit isoform of Na+,K(+)-ATPase and 2) raise the possibility that the previous finding of a mutation in the cDNA of the SS/Jr rat may have been due to a reverse transcriptase error during cDNA synthesis.

The rat renin gene: assignment to chromosome 13 and linkage to the regulation of blood pressure.

It has recently been suggested that in the rat, sequence variation in the renin gene or closely linked genes may have the capacity to affect blood pressure and contribute to the pathogenesis of hypertension. To map the chromosomal location of the rat renin gene and to investigate its relationship to the inheritance of increased blood pressure, we studied a panel of rat x mouse somatic cell hybrids and a large set of recombinant inbred (RI) strains derived from spontaneously hypertensive rats (SHR) and normotensive Brown-Norway (BN) rats. We have found that in the rat, the renin gene is located on chromosome 13 and that it belongs to a conserved synteny group located on chromosome 1 in man and mouse. We have also found the median blood pressure of the RI strains that inherited the renin allele of the SHR to be greater than that of the RI strains that inherited the renin allele of the normotensive BN rat. These findings, together with the results of previous studies, suggest that in the rat, sequence variation in the renin gene, or in genes linked to the renin locus on chromosome 13, may have the capacity to affect blood pressure.

Cosegregation of blood pressure with a kallikrein gene family polymorphism.

It has recently been proposed that sequence variation in the gene coding for tissue kallikrein might be involved in the pathogenesis of hypertension. However, molecular evidence of an association between a sequence alteration in the kallikrein gene family and the transmission of increased blood pressure has never been reported. In 32 recombinant inbred (RI) strains derived from the spontaneously hypertensive rat (SHR) and the normotensive Brown Norway rat (BN), we investigated whether a restriction fragment length polymorphism (RFLP) marking the kallikrein gene family cosegregated with blood pressure. In the RI strains that inherited the kallikrein RFLP from the SHR progenitor strain, the median systolic, diastolic, and mean arterial pressures were significantly greater than in the RI strains that inherited the kallikrein RFLP from the BN progenitor strain. These findings suggest that in the rat, sequence variation in the kallikrein gene family, or in closely linked genes, may have the capacity to affect blood pressure.

Biometric genetic analysis of blood pressure in the spontaneously hypertensive rat.

The spontaneously hypertensive rat is the most widely studied animal model of essential hypertension, yet the genetics of transmission of high blood pressure in this strain have not been clearly defined. It has been proposed that in the spontaneously hypertensive rat, blood pressure follows a simple additive mode of inheritance and that the hypertension is primarily determined by a single major locus. To investigate the genetics of transmission of increased blood pressure in the spontaneously hypertensive rat, we performed a biometric genetic analysis of multiple, direct measurements of arterial pressure in unanesthetized, unrestrained rats derived by crossing spontaneously hypertensive rats with two different inbred normotensive strains, the Charles River Wistar-Kyoto rat and the Lewis rat. In both crosses, approximately 60% of the variation in blood pressure could be assigned to genotypic variation. The data fit an additive-dominance model of inheritance in which alleles decreasing blood pressure were partially dominant. Thus, in offspring derived from crosses between spontaneously hypertensive rats and Wistar-Kyoto rats or spontaneously hypertensive rats and Lewis rats that are raised under ordinary laboratory conditions, increased blood pressure is not determined by simple additive effects of alleles at a single major locus. The current findings are consistent with the possibility that in the spontaneously hypertensive rat, hypertension may arise from mutations in alleles that ordinarily act in a dominant fashion to suppress blood pressure.

Cosegregation of the renin allele of the spontaneously hypertensive rat with an increase in blood pressure.

The spontaneously hypertensive rat (SHR) exhibits alterations in the renin-angiotensin-aldosterone system which are similar to those that characterize patients with "nonmodulating" hypertension, a common and highly heritable form of essential hypertension. Accordingly, we determined whether the inheritance of a DNA restriction fragment length polymorphism (RFLP) marking the renin gene of the SHR was associated with greater blood pressure than inheritance of a RFLP marking the renin gene of a normotensive control rat. In an F2 population derived from inbred SHR and inbred normotensive Lewis rats, we found the blood pressure in rats that inherited a single SHR renin allele to be significantly greater than that in rats that inherited only the Lewis renin allele. To the extent that the SHR provides a suitable model of "nonmodulating" hypertension, these findings raise the possibility that a structural alteration in the renin gene, or a closely linked gene, may be a pathogenetic determinant of increased blood pressure in one of the most common forms of essential hypertension in humans.