Biotechnology. Paper Alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in biotechnology.     

Effects of the plasticiser DEHP on lung of newborn rats: catalase immunocytochemistry and morphometric analysis

Experimental administration of di-(2-ethylexyl)-phthalate (DEHP), a plasticiser employed in the fabrication of polyvinyl chloride (PVC), causes increases in lipid metabolising enzymes along with marked peroxisomal proliferation. The effects are found in several mammalian tissues, of which the rodent liver is the most responsive target. Leakage of DEHP from PVC devices is favoured by high temperature and contact with lipid-containing biological fluids. Since preterm babies are currently ventilated through endotracheal PVC tubes, it seemed worthwhile to investigate DEHP effects on immature mammalian lung. In this research, female rats were fed with DEHP in the last week of pregnancy and after delivery, and lungs were excised from 2-day-old pups. At this age, in fact, rat lung histological features closely resemble those found in 24- to 36-week-old human fetuses. In treated animals, morphometric analysis of histological parameters revealed a dramatic decrease in the number of parenchymal airspaces, together with significant increases in their mean size. Moreover, cytochemical detection of the peroxisomal marker catalase revealed an increase in the number of type II pneumocytes. Our findings closely resemble abnormal histological features observed in autoptic lung specimens from children affected with chronic lung diseases.     

Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.     

Replication restart in gyrB Escherichia coli mutants

Gyrase is an essential topoisomerase in bacteria that introduces negative supercoils in DNA and relaxes the positive supercoils that form downstream of proteins tracking on DNA, such as DNA or RNA polymerases. Two gyrase mutants that suffer partial loss of function were used here to study the need for replication restart in conditions in which gyrase activity is affected. We show that the preprimosomal protein PriA is essential for the viability of these gyrB mutants. The helicase function of PriA is not essential. The lethality of the gyrB priA double mutants is suppressed by a dnaC809 mutation, indicating a requirement for primosome assembly in gyrB strains. The lethality of gyrB priA combination of mutations is independent of the level of DNA supercoiling, as gyrB and priA were also co-lethal in the presence of a DeltatopA mutation. Inactivation of homologous recombination did not affect the viability of gyrB mutants, indicating that replication restart does not require the formation of a recombination intermediate. We propose that the replisome is disassembled from replication forks when replication progression is blocked by the accumulation of positive supercoils in gyrase mutants, and that replication restarts via PriA-dependent primosome assembly, directly on the in-activated replication forks, without the formation of a recombination intermediate.     

Development and characterization of secretin-stimulated secretion of cultured rat cholangiocytes

We sought to develop a cholangiocyte cell culture system that has preservation of receptors, transporters, and channels involved in secretin-induced secretion. Isolated bile duct fragments, obtained by enzyme perfusion of normal rat liver, were seeded on collagen and maintained in culture up to 18 wk. Cholangiocyte purity was assessed by staining for gamma-glutamyl transpeptidase (gamma-GT) and cytokeratin-19 (CK-19). We determined gene expression for secretin receptor (SR), cystic fibrosis transmembrane conductance regulator, Cl(-)/HCO(3)(-) exchanger, secretin-stimulated cAMP synthesis, Cl(-)/HCO(3) exchanger activity, secretin-stimulated Cl(-) efflux, and apical membrane-directed secretion in polarized cells grown on tissue culture inserts. Cultured cholangiocytes were all gamma-GT and CK-19 positive. The cells expressed SR and Cl(-)/HCO(3)(-) exchanger, and secretin-stimulated cAMP synthesis, Cl(-)/HCO(3)(-) exchanger activity, and Cl(-) efflux were similar to freshly isolated cholangiocytes. Forskolin (10(-4) M) induced fluid accumulation in the apical chamber of tissue culture inserts. In conclusion, we have developed a novel cholangiocyte line that has persistent HCO(3)(-), Cl(-), and fluid transport functions. This cell system should be useful to investigators who study cholangiocyte secretion.     

N-benzhydryl-glycolamide: the first protecting group in peptide synthesis with a strong conformational bias

We have synthesized and examined the preferred conformation of a set of N-benzhydryl-glycolamide esters from N(alpha)-protected (or N(alpha)-blocked) alpha-amino acids. Experiments were performed in CDCl(3) solution by Fourier transform infrared absorption and (1)H-NMR techniques, and in the crystalline state by x-ray diffraction. The results of our analysis strongly support the view that this type of N(alpha)-acylated alpha-aminoacyl esters has a marked tendency to fold into a beta-turn conformation, the nature of which is dictated by the structural propensity of the amino acid constituent at the i+1 position.     

Conformational constraints of tyrosine in protein tyrosine kinase substrates: Information about preferred bioactive side-chain orientation

The side-chain orientation of a tyrosine residue located in a peptide, which is an excellent substrate of Syk tyrosine kinase (A. M. Brunati, A. Donella-Deana, M. Ruzzene, O. Marin, L. A. Pinna, FEBS Letters, 1995, Vol. 367, pp. 149-152), was fixed in the gauche (+) or gauche (-) conformation by using the 7-hydroxy-1,2,3,4-tetrahydro isoquinoline-3-carboxylic (Htc) structure. The tyrosine trans conformation was blocked by using an aminobenzazepine-type (Hba) structure. The proposed side-chain orientations were confirmed by the analysis of the (1)H-NMR parameters: chemical shifts, coupling constants, and nuclear Overhauser effects to the tyrosine constraints in the different analogs. This "rotamer scan" of the phosphorylatable residue allowed us to generate optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase. In contrast, these conformationally restricted tyrosine analogs were not tolerated by the Src-related tyrosine kinases Lyn and c-Fgr.     

Observations on Rhinomonas reticulata comb. nov. and R. reticulata var. eleniana var. nov. (Cryptophyceae), with comments on the genera Pyrenomonas and Rhodomonas

Seven marine red strains of Cryptophyceae were examined using scanning and transmission electron microscopy. Six of these, possessing a nucleomorph housed within the pyrenoid and a periplast with distinctly hexagonal periplast areas, are assigned to Rhinomonas reticulata comb. nov. Rhinomonas reticulata var. eleniana var. nov. is described on the basis of the smaller size of the periplast areas. In a strain of the genus Pyrenomonas , which Rhinomonas otherwise resembles, the periplast areas appear more or less rectangular. Since it is impossible to know with certainty which red marine forms fall within the original concept of the genus Rhodomonas , the name Pyrenomonas should be preferred to a recent emendation of Rhodomonas.