Relationship between cell density and prolidase activity in human skin fibroblasts: effects of ascorbate and fructose

To evaluate the influence of cell density on the activity of fibroblast prolidase (EC 3.4.13.9), we determined this activity in sparse and dense cultures. We also investigated, the effects of different concentrations of β-d(?) fructose and l(+) ascorbate, which both increased cell density at confluency. For a fructose concentration of 25 mM, we observed that in the absence of glucose, intracellular total proteins increased 1.5-fold and prolidase specific activity, 1.8-fold. For ascorbate, a broad optimum concentration was found (range 0.01 – 0.50 mM). Addition to cultures of 0.1 mM ascorbate increased total proteins 1.4-fold, and doubled prolidase activity. This investigation was prompted by our previous results [J. Metab. Dis. 1983, 6, 27–31], confirmed here, and suggesting that increased prolidase activity at confluency was due to a rise in cell density.     

Alpha-DNA. IV: Alpha-anomeric and beta-anomeric tetrathymidylates covalently linked to intercalating oxazolopyridocarbazole. Synthesis, physicochemical properties and poly (rA) binding

A new set of molecules made of an intercalating agent (oxazolopyridocarbazole, OPC) covalently linked through a polymethylene chain of various length to the 3' end of alpha-anomeric or beta-anomeric tetradeoxynucleotides (alpha- or beta-T4) have been synthesized. The beta-thymidylate modified compound (beta-T4C5OPC) is able to interact with the complementary sequence, beta-poly (rA); this interaction is strongly stabilized compared to the parent compound, beta-oligo(dT)4 and is specific for poly (rA). The molecule synthesized from the unnatural alpha-anomer, alpha-T4C5OPC, is also able to interact with poly (rA) leading to the formation of an alpha-beta hybrid stabilized by the energy provided by the OPC moiety. The stoechiometry of the binding reaction shows that an A-T pairing occurs in the alpha-beta heterohybrids. Tm studies reveal that the alpha-beta heterohybrids are more stable than their beta-beta counterparts.     

The compositional distribution of coding sequences and DNA molecules in humans and murids

Summary The compositional distributions of coding sequences and DNA molecules (in the 50-100-kb range) are remarkably narrower in murids (rat and mouse) compared to humans (as well as to all other mammals explored so far). In murids, both distributions begin at higher and end at lower GC values. A comparison of homologous coding sequences from murids and humans revealed that their different compositional distributions are due to differences in GC levels in all three codon positions, particularly of genes located at both ends of the distribution. In turn, these differences are responsible for differences in both codon usage and amino acids. When GC levels at first+second codon positions and third codon positions, respectively, of murid genes are plotted against corresponding GC levels of homologous human genes, linear relationships (with very high correlation coefficients and slopes of about 0.78 and 0.60, respectively) are found. This indicates a conservation of the order of GC levels in homologous genes from humans and murids. (The same comparison for mouse and rat genes indicates a conservation of GC levels of homologous genes.) A similar linear relationship was observed when plotting GC levels of corresponding DNA fractions (as obtained by density gradient centrifugation in the presence of a sequence-specific ligand) from mouse and human. These findings indicate that orderly compositional changes affecting not only coding sequences but also noncoding sequences took place since the divergence of murids. Such directional fixations of mutations point to the existence of selective pressures affecting the genome as a whole.     

Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation.

Using a polyclonal antibody raised against B2 cyclin from Xenopus laevis, we show that prophase-arrested Xenopus oocytes contain a stockpile of cyclin B2 protein. During progesterone-induced maturation, an increase in the synthesis of cyclin B2 is observed, although Western blotting experiments show that this new synthesis does not significantly increase the mass of cyclin over the maternal stockpile. In the oocyte cyclin B2 is already present in two forms which differ in the extent of phosphorylation, but the phosphorylated form becomes predominant as oocytes progress towards germinal vesicle breakdown (GVBD), coincident with cdc2 protein kinase activation. These two events do not depend upon formation of a new complex between cyclin and cdc2 protein kinase, since these two proteins are already found associated in resting oocytes, prior to activation of the kinase.     

Poly(rA) binding of alpha-anomeric and beta-anomeric tetrathymidylates covalently linked to an intercalating oxazolopyridocarbazolium. Determination of the binding parameters

We have investigated by means of absorbance measurements at 310 nm the binding of alpha-anomeric or beta-anomeric tetrathymidylates covalently substituted at their 3' end by an intercalating agent (oxazolopyridocarbazolium), to poly(rA). Taking into account the strong autoaggregation of the free ligands, we have derived the binding parameters corresponding to the [alpha] and the [beta] ligands. The affinity of the alpha-anomer for poly(rA) is higher than the affinity of the beta-anomer in accordance with the Tm studies conducted on such a system.     

Two Down syndrome patients with rec(21),dupq,inv(21)(p11;q2109) from a familial pericentric inversion

Aneusomie de recombinaison arose from a familial pericentric inversion of a chromosome 21. Two female patients had a typical Down syndrome; one of them had slight psychomotor retardation. There was partial trisomy 21q2109----qter in these two patients but ZnCu SOD activity was normal.     

Hypoxia-induced changes in shivering and body temperature

Experiments were carried out on conscious cats to evaluate the general characteristics and modes of action of hypoxia on thermoregulation during cold stress. Intact and carotid-denervated (CD) conscious cats were exposed to ambient hypoxia (low inspired O2 fraction) or CO hypoxia in prevailing laboratory (23-25 degrees C) or cold (5-8 degrees C) environments. In the cold, both groups promptly decreased shivering and body temperature when exposed to either type of hypoxia. Small increases in CO2 concentration reinstituted shivering in both groups. At the same inspired concentration of O2, CD animals decreased shivering and body temperature more than intact cats. While this difference resulted, in part, from a lower alveolar PO2 in CD cats, a difference between intact and CD cats was apparent when the two groups were compared at the same alveolar PO2. During more prolonged hypoxia (45 min), shivering returned but did not reach normoxic levels, and body temperature tended to stabilize at a hypothermic value. Exposure to various levels of hypoxia produced graded suppression of shivering, with the result that the change in body temperature varied directly with inspired O2 concentration. Hypoxia appears to act on the central nervous system to suppress shivering and sinus nerve afferents appear to counteract this direct effect of hypoxia. In intact cats, this counteraction appears to be sufficient to maintain body temperature under hypoxic conditions at room temperature but not in the cold.     

L'ültrastructure du Foie Humain Lors D'ictères Idiopathiques Chroniques

Summary 15 liver biopsies from 13 patients suffering from non-hemolytic constitutional jaundice (6 cases of Gilbert's, 2 cases of Rotor's and 5 cases of Dubin-Johnson's disease) provided the basis of an ultrastructural study of pigment inclusions in liver cells following various techniques of preparation.The lipofuscin in Gilbert's syndrom and the pigment characteristic of Dubin-Johnson's syndrom contain two distinct components which are present in both types of inclusions but their proportions are different in these syndromes: a globular component, probably of lipidic nature, is the main constituent of the lipofuscin, a granulo-filamentous component the main constituent of the Dubin-Johnson's pigment. These two components react in both syndromes identically to various techniques of fixation, embedding and section-staining. In Rotor's syndrome other pigment inclusions are observed which are not detectable in ordinary histological preparations: they consist essentially of aggregates of dense, ferritin-like material. The presence, in the hepatocytes, of pigment inclusions which have some features in common in the 3 types of syndromes and other characteristics specific for each of them, confirms the close relation between these types of jaundice, but emphasizes also their differences; moreover, this ultrastructural study underlines the difficulty of an exact classification of these syndromes.

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Travail subventionné par la Fondation Fritz Hoffmann-La Roche pour l'expansion en Suisse du travail scientifique exécuté par équipe et par le Fonds National Suisse de la Recherche Scientifique.

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Avec la collaboration technique de Mlle M.-M. Bertholet.     

Role of dopamine and arterial chemoreceptors in thermal tachypnea in conscious cats

In mammals submitted to a warm environment, intracerebral injection of dopamine (DA) produces no change or an increase in body temperature accompanied by an increase in metabolic heat production, but its effect on heat loss mechanisms such as vasodilation and tachypnea is not clear. Because the principal mechanism of heat loss in the conscious cat is thermal tachypnea, we studied the influence of DA on thermal tachypnea in response to heat stress (ambient temperature = 33-36 degrees C) in five conscious cats. We first studied the steady-state response to a DA agonist, apomorphine, which crosses the blood-brain barrier. Intravenous injection of apomorphine greatly reduced thermal tachypnea by decreasing respiratory frequency (from 94.9 to 52.5 breaths/min) and increasing tidal volume (from 13.2 to 20.4 ml). The subsequent injection of the DA antagonist haloperidol, which also crosses the blood-brain barrier, restored the initial tachypnea. To further investigate the mechanism involved in thermal tachypnea, we studied the influence of peripheral chemoreceptors by transiently stimulating or inhibiting carotid body (CB) activity during tachypneic breathing. CB stimulation by intravenous injection of NaCN or domperidone reduced thermal tachypnea mainly by decreasing the respiratory frequency, whereas CB inhibition by DA tended to increase frequency and thus tachypnea. It is concluded that 1) in a warm environment, central DA receptors are also greatly involved in heat loss mechanisms, 2) arterial chemoreceptor input appears to counteract this tachypneic breathing, and 3) thermal and hypoxic tachypnea may be controlled by the same mechanism in which a DA-like system has a key role.     

A large multigene family codes for the polypeptides of the crystalline trichocyst matrix in Paramecium.

The secretory granules (trichocysts) of Paramecium are characterized by a highly constrained shape that reflects the crystalline organization of their protein contents. Yet the crystalline trichocyst content is composed not of a single protein but of a family of related polypeptides that derive from a family of precursors by protein processing. In this paper we show that a multigene family, of unusually large size for a unicellular organism, codes for these proteins. The family is organized in subfamilies; each subfamily codes for proteins with different primary structures, but within the subfamilies several genes code for nearly identical proteins. For one subfamily, we have obtained direct evidence that the different members are coexpressed. The three subfamilies we have characterized are located on different macronuclear chromosomes. Typical 23-29 nucleotide Paramecium introns are found in one of the regions studied and the intron sequences are more variable than the surrounding coding sequences, providing gene-specific markers. We suggest that this multigene family may have evolved to assure a microheterogeneity of structural proteins necessary for morphogenesis of a complex secretory granule core with a constrained shape and dynamic properties: genetic analysis has shown that correct assembly of the crystalline core is necessary for trichocyst function.