Melanin as a natural germ cell marker for intraspecific transplantation experiments in Ambystoma mexicanum (Urodela,Amphibia)

Summary In urodele amphibians, the lack of a reliable germ cell marker restricts the experimental study of the germ lineage. In the present work, we conducted genetic and histological analyses in order to demonstrate that melanin from oocytes constitutes a germ cell marker available for intraspecific experiments in Ambystoma mexicanum. Then, using this marker, we implanted germ cells from undifferentiated gonads (stage 48) into the blastocoel of host embryos and investigated their fate and determined state. Our results show that, from this stage on, the donor cells do not differentiate into other cell types; therefore, they are restricted in developmental capacity and irreversibly determined as germ cells. On the other hand, exogenous germ cells were found in an isotopic position until the young tail-bud stage, and then were found in an ectopic position; these results suggest that, from the middle tail-bud stage on, an active process contributes to migration of primordial germ cells to the gonadal territory.     

Bacterial siderophores : structure and NMR assignment of pyoverdins Pa,siderophores ofPseudomonas aeruginosa ATCC 15692

Summary In iron-deficient conditions,Pseudomonas aeruginosa ATCC 15692 synthesizes two major siderophores, pyoverdins Pa and pyoverdin Pa B. Two other compounds, pyoverdin Pa A (occurring from hydrolysis of pyoverdin Pa during the culture) and pyoverdin Pa C (occurring artifactually during the purification procedure) were also isolated. All these compounds possess the same partly cyclic peptide chain wherel-Orn(OH · HCO) isN -formyl,N -hydroxy-l-ornithine. The chain is bound to a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and having the (S) configuration. The four pyoverdins differ only in the acyl substituent bound to the nitrogen atom bound to carbon C3 of the chromophore. This is succinamide (pyoverdin Pa), succinic acid (pyoverdin Pa A), methyl succinate (pyoverdin Pa C) and 2-oxoglutaric acid (pyoverdin Pa B). The complete¹H- and¹³CNMR assignments, using two-dimensional total correlation NMR spectroscopy (TOCSY) and rotating-frame Overhauser enhancement spectroscopy (ROESY) procedures, as well as¹H-¹³C correlations, are reported. The complete sequence of the peptide using CH-NH correlations was achieved by NMR and confirmed the partly cyclic structure earlier reported using fast-atom-bombardment mass spectrometry (FAB-MS) on the siderophores and their dansylated fragments [Briskot G, Taraz K, Budzikiewicz H (1989)Liebigs Ann Chem: 375–384]. The use of these NMR procedures appears to be a tool of choice and a complementary approach to FAB-MS in the structure determination of some complex pyoverdins.Abbreviations Ser serine - Arg arginine - Thr ethreonine - Lys lysine - OHOrn N -hydroxyornithine - Chr chromophore     

Control of myogenesis in the mouse myogenic C2 cell line by medium composition and by insulin: Characterization of permissive and inducible C2 myoblasts

Using subcloning and manipulations of culture conditions we have isolated from the mouse myogenic cell line C2 a variant cell line that we named inducible. Unlike the progenitor cells that are referred to as permissive, inducible myoblasts differentiate poorly in Dulbecco modified Eagle medium plus fetal calf serum (FCS) and require the presence of insulin at a high concentration (1.6 10(-6) M) or insulin-like growth factor I (IGFI) at a lower concentration (2.5 10(-8) M) to differentiate. Permissive and inducible myoblasts fail to differentiate when grown in MCDB202 medium plus 20% FCS, even after a prolonged arrest in G1 phase. This shows that an arrest in G1 is in itself insufficient to trigger terminal differentiation. Both cell types also exhibit distinct patterns of accumulation of muscle mRNAs corresponding to sarcomeric actins and myosin light chain MLC1A. The possibility that these two cell lines might represent two different stages of the progression of myoblasts toward terminal differentiation is discussed.     

Antigenic determinants expressed by human C4 allotypes; a study of 325 families provides evidence for the structural antigenic model

The antigenic determinants of human C4 have been defined by human IgG antisera, Rodgers (Rg) and Chido (Ch), in hemagglutination-inhibition assays (HAI). Eight (2 Rg and 6 Ch) are of high frequency, > 90% , and 1, WH, is of low frequency, 15 %. The phenotypic combinations are complex; generally, C4A expresses Rg, and C4B has Ch, but reverse antigenicities have been established both by HAI and by sequence data of selected C4 allotypes. A study of 325 families provides data on the antigenic expression of each C4 allotype and demonstrates strong associations. A structural model for the antigenic determinants of C4 proteins has been proposed and is completely supported by the family material. Of the 16 possible antigenic combinations for C4 proteins, only 3 are undetected. A new Ch combination has been recorded in two French families. The reported sequence variation within the C4d region can account for the antigenic determinants but leaves the location of electrophoretic variation in C4 still unclear.     

Optimization of the spore production of Penicillium roqueforti in solid substrate fermentation on buckwheat seeds

Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.10⁹ spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 10⁹ spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media.     

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.     

Interrupter resistance elucidated by alveolar pressure measurement in open-chest normal dogs

The interrupter method for measuring respiratory system resistance involves rapidly interrupting flow at the mouth while measuring the pressure just distal to the point of interruption. The pressure signal observed invariably exhibits two distinct phases. The first phase is a very rapid jump, designated delta Pinit, which occurs immediately on interruption of flow. The second phase is designated delta Pdif and is a further pressure change in the same direction as delta Pinit but evolving over several seconds. The physiological interpretations of delta Pinit and delta Pdif have been somewhat unclear. Delta Pinit has been taken to equal the pressure drop across the pulmonary airways, possibly with a contribution from the tissues of the respiratory system. Delta Pdif can arise, in principle, from two sources: gas redistribution throughout the lung after interruption of flow and stress recovery within the tissues. To resolve these issues we performed interruption experiments on anesthetized paralyzed, tracheotomized, open-chest normal dogs during passive expiration while measuring alveolar pressures at three sites with alveolar capsules. We found that, in the absence of the chest wall, delta Pinit reflects only the resistance of the airways and that delta Pdif can be ascribed almost entirely to the stress recovery properties of lung tissues.     

Circadian and Ultradian Rhythms in Blood Glucose and Plasma Insulin of Healthy Adults

The circadian and ultradian variations of blood glucose and plasma insulin have been characterized individually and as a group phenomenon in five healthy young adults studied while adhering as closely as possible to their usual routine of sleep, activity, meal content and timing. Three complementary methods were used to analyze the data: displaying raw data as a function of time; cosinor method according to Nelson and Halberg; and time series analyses as proposed by De Prins and Malbecq. The subjects were studied in the laboratory and their life routine were controlled, but very close to that of their habitual routine. They had mainly ultradian rhythms of blood glucose (mainly about 6 hr) and circadian rhythms of immunoreactive insulin (I.R.I.). Blood glucose ultradian rhythms seem to be mainly but not exclusively mealtime dependent, while I.R.I, circadian rhythms appear to be primarily endogenous in origin. Therefore, the role played by insulin in the control of blood glucose levels seems to be programmed on a circadian basis rather than by a time independent feedback phenomenon as postulated by the conventional homeostatic hypothesis. The advantage of this chronophysiologic approach is to consider circadian rhythms of both I.R.I. and insulin effectiveness as an adaptive phenomenon able to maintain blood sugar changes in the ultradian domain of rhythms.     

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Endogenous cholesterol synthesis is sufficient for ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture, fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented medium. This work was supported by Finnish Culture Foundation.