L-proline uptake in human fibroblasts: evidence for a high-affinity system in addition to system A

Proline uptake was studied in human skin fibroblasts by simultaneous running of kinetic and inhibition experiments on the same cell lines. Two systems for proline uptake were shown: a high-affinity system not inhibited by alpha-(methylamino)isobutyric acid and a low affinity system inhibited by this amino acid (i.e. system A). These results appear to be of interest, firstly because up till now, system A was considered preferable for proline uptake in human fibroblasts, and secondly because they illustrate the need for combined inhibition and kinetic studies of amino acid uptake, especially when the substrate concentration range used and the respective Km of the systems do not allow their detection by kinetic analysis alone. Furthermore, this high-affinity system may have major physiological implications.     

Evidence for the activation of phospholipases during acrosome reaction of human sperm elicited by calcium ionophore A23187

Incubation of washed human sperm with [3H]- or [14C]arachidonic acid allowed a major incorporation of the label into phospholipids, provided that the final concentration of the fatty acid did not exceed 20 microM. A further challenge with calcium ionophore A23187 of spermatozoa suspended in a calcium-containing medium led to phospholipid hydrolysis, which could account for 10-12% of total cell radioactivity. Degradation products were identified as free, unconverted arachidonic acid, occurring with some diacylglycerol. Phospholipid hydrolysis was significant after 15 min of incubation and became maximal after 120 min. It was found to be calcium dependent, diacylglycerol and free arachidonate production occurring maximally at 2 mM and 5 mM CaCl2, respectively. Phosphatidylcholine and phosphatidylinositol were the most significantly degraded phospholipids after 60 min of incubation. Similar incubations conducted with 32P-labeled sperm confirmed the selective hydrolysis of phosphatidylcholine and revealed an increase production of phosphatidic acid probably due to a phosphorylation of diacylglycerol. Under the same conditions, one third of the cells remained motile and electron microscopy revealed that acrosome reaction was completed in 40% of the cells and displayed an intermediary state in 40-50% of the spermatozoa. Furthermore, a good parallelism was observed between the extent of the acrosome reaction and the extent of phospholipid hydrolysis promoted by increasing concentrations of A23187. It is concluded that calcium entry into the cells activates both a phospholipase A2 and a phospholipase C, leading to the production of substances, like lysophospholipid, diacylglycerol or phosphatidic acid, which may or may not be involved in acrosome reaction.     

Factors influencing genetic counseling attendance rate: a geographically based study

A study of factors influencing genetic counseling attendance rate has been conducted in the Bouches-du-Rh?ne area, in the south of France. In this area, a birth defects monitoring system (Eurocat n. 22) annually covers 23,000 births. All the genetic services are delivered by only one genetic center located in Marseilles, and the data collected are computerized. The comparison of these two data bases gives an opportunity to estimate the rate of genetic counseling attendance after the occurrence of an affected stillbirth or live birth. Among the parents of 358 infants born in 1983-84 in this area with a pathology requiring genetic counseling, 226 (63 per cent) attended the Genetic Center within the first year after birth. The rate of attendance is statistically higher (p less than 0.01) for the parents who had a stillbirth (78 per cent) than for those who had a live birth (57 per cent). It is also higher (p less than 0.01) for the Marseilles maternities group (68 per cent) than for the group outside Marseilles (50 per cent). The referral delays are also analyzed according to malformation etiology and to viability of the child by the eighth day of life. Besides evaluating a particular genetic center's effectiveness in diffusing information to the public concerned, this work shows that couples' request is strongly dependent on a psychological need.     

Amino acid transport systems in the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Evidence that cycloleucine affects the high-affinity systems of amino acid uptake in cultured human fibroblasts.

The influence of cycloleucine on kinetic parameters of uptake of L-alanine, L-proline and L-leucine into cultured human fibroblasts was examined under initial-rate conditions with substrate concentrations of 0.05-10 mM and 5 mM-cycloleucine. Kinetic data obtained by computer analysis showed that, in the absence of cycloleucine, cell uptake was heterogeneous for each amino acid. L-Alanine and L-leucine entered by two transport systems with different affinities; L-proline was taken up by one saturable transport system plus a diffusion-like process. This heterogeneity disappeared in the presence of cycloleucine, since the high-affinity systems were no longer detectable. The remaining process had the same kinetic constants as the low-affinity system for alanine and leucine and a KD similar to the diffusion constant for proline. The influence of cycloleucine on the amino acid uptake was not specific either to the amino acid concerned or to a particular transport system, since the three neutral amino acid-transport systems, A, ASC and L, were involved in these experiments. This influence was shown to be unaffected by the absence of Na+ (for leucine uptake). ATP content of the cells was identical in the presence or in the absence of cycloleucine.     

Fractionation of charge-modified low density lipoproteins by fast protein liquid chromatography.

We describe a methodology developed to separate different forms of charge-modified low density lipoproteins (LDL) using the fast protein liquid chromatography (FPLC) system from Pharmacia. Lipoproteins were isolated by sequential ultracentrifugation and introduced onto an anion-exchange column (Mono Q HR 5/5). The multistep NaCl gradient elution was optimized and the analytical variables were determined on copper-oxidized LDL. After oxidation by copper for various times (up to 48 h), five forms were obtained (fractions A, B, C, D, and E). Within-run and day-to-day reproducibility were better than 8.6% and 10%, respectively. Protein and cholesterol recovery after the chromatographic separation was good (greater than 82%) and the detection limit was about 1 microgram. The more negative forms of collected LDL were mainly characterized by an increase in the lipid peroxidation product content, a depletion of vitamin E, an alteration of apoB and increased degradation by macrophages. The proposed methodology was applied to the study of LDL modifications generated by human umbilical endothelial cells and the protective effect of antioxidants (vitamin E and probucol).     

What can we learn from noncoding regions of similarity between genomes?

Background  

In addition to known protein-coding genes, large amounts of apparently non-coding sequence are conserved between the human and mouse genomes. It seems reasonable to assume that these conserved regions are more likely to contain functional elements than less-conserved portions of the genome.