Thymic nurse cells: morphological study during their isolation from murine thymus

Summary Thymic nurse cells (TNC), which are multicellular complexes composed of epithelial cells and thymocytes, were obtained from C3H-mice thymuses. They were described by means of light and electron microscopy. The morphology of epithelial cells forming isolated TNC compared to that of small tissue fragments obtained by enzymatic digestion revealed that TNC could be derived from all parts of the thymus: cortex, corticomedullary junction and medulla, the cortex being their principal source. This variety of origin, the presence of several epithelial cells inside a single TNC, the presence of non-lymphoid cells, and the various locations of eleaved desmosomes confirmed that their aspect in vitro as round and sealed structures can be considered to be an artifact due to the isolation technique used. Indeed, during this procedure, they are formed by a process of wrapping of the epithelial cytoplasm around the tightly associated thymocytes. All three epithelial cell types: cortical reticular cells, medullary reticular cells, and medullary globular cells can form TNC.A portion of this work was presented at the first Thymus Workshop. Rolduc, Netherlands, April, 1988     

Ketone Body Utilization for Lipid Synthesis in the Murine Sciatic Nerve: Alterations in the Dysmyelinating Trembler Mutant

This work demonstrates that in vitro sciatic nerves of normal and trembler adult mice can use ketone bodies (beta-hydroxybutyrate and acetoacetate) and butyrate for lipid synthesis. In normal sciatic nerves, beta-hydroxybutyrate is incorporated in total lipids to a larger extent than acetoacetate (141% and 33%, respectively, of acetate incorporation), whereas for trembler sciatic nerves, these percentages are only 69% and 27%. Incorporation of ketone bodies is greater into sterols than into other lipids. Lipid metabolism of ketone bodies in trembler nerves is altered and could reflect a process similar to Wallerian degeneration: a dramatic decrease of sterol and free fatty acid synthesis and an increased synthesis of triglycerides. Moreover, differences seen in precursor incorporation into lipids between normal and trembler sciatic nerves suggest that their lipid metabolism is not the same.     

A novel sickle cell mutation of yet another origin in Africa: the Cameroon type

Summary The sickle cell mutation (^s) arose as at least three independent events in Africa and once in Asia, being termed the Senegal, Benin, Bantu and Indian types respectively. An investigation in Cameroon was carried out to determine whether the atypical sickle genes observed in the neighboring countries are the result of recombination or the presence of a sickle cell mutation of a different genetic origin. It was conducted on 40 homozygous SS patients followed at the Blood Transfusion Center in the capital city of Yaoundé. On 80 ^s chromosomes, 13 exhibited a novel polymorphic pattern that was observed three times in the homozygous state. This chromosome contains an ^AT gene. The restriction fragment length polymorphism haplotype is different from all the other ^s chromosomes in both the 5 and 3 regions, but has previously been reported in sporadic cases. The (AT)8(T)5 sequence in the — 500 region of the gene is specific and different from that of the Senegal, Benin, Bantu or Indian ^s genes. All the carriers of this specific chromosome belong to the Eton ethnic group and originate from the Sanaga river valley. This observation strongly argues for yet another independent origin of the sickle cell mutation in Africa, here referred to as the Cameroon type. The Benin haplotype and a Benin/ Bantu recombinant haplotype have been observed in the other studied populations: Ewondo, Bamiléké, Bassa, Yambassa and Boulou.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France

RFLPs of 68 normal and 74 mutant alleles at the phenylalanine hydroxylase (PAH) locus were determined in 37 French kindreds. A total of 23 haplotypes, including 18 normal and 16 mutant alleles, were observed. Two-thirds of all mutant alleles were confined within only four haplotypes, while the last third was accounted for by 12 haplotypes, including eight haplotypes absent from Caucasian pedigrees reported thus far. Several mutant haplotypes were present in typical phenylketonuria only, others were present in variants only, and some were present in both. In addition, a particular mutant haplotype (haplotype 2) was found to harbor different mutations in our series, resulting in either typical phenylketonuria or in mild hyperphenylalaninemias. The diploid combination of so many mutant haplotypes in PAH-deficient patients and of compound heterozygosity at the PAH locus in southern Europe might account for the broad spectrum of individual phenotypes observed in France.     

Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein

Based on sequence information from tryptic peptides an almost full-size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced. The coding region was cloned into an Escherichia coli expression vector and the protein expressed at high levels. The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays.     

Resumption of cellular activity induced by cytokinin and gibberellin treatments in tomato flowers targeted for abortion in unfavorable light conditions

Mitotic activity and nuclear DNA synthesis in tomato ( Lycopersicon esculentum Mill., cv. King plus) flowers targeted for abortion under unfavorable light conditions are completely stopped 6 days after macroscopic appearance of the inflorescence. Ovular cells are arrested at the G₁ (80%) and G₂ (20%) stages of the cell cycle. Exogenous applications of a mixture of N⁶-benzyladenine (BA) and gibberellins A₄₊₇ (GA) directly on the inflorescence may prevent its failure. Nuclear DNA synthesis and mitoses resume in ovules of the flower 16 to 20 h after the BA+GA treatment. When applied alone, BA and GA are able to mimic the effect of the mixture upon the progression of ovular cells through their cycle. Sporogenesis processes are also set in motion by the exogenous plant growth regulators. The mechanism of action of cytokinins and gibberellins in the control of floral development is discussed.     

Myelin-associated glycoprotein and myelin basic protein are present in central and peripheral nerve myelin throughout phylogeny

This phylogenetic study of central and peripheral nervous system myelin proteins demonstrates that important changes occur in the composition of certain myelin proteins during evolution. Only two components, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) are present in all Gnathostomata representatives investigated. While MBP components varied considerably even among the representatives of a given order, the apparent molecular weight of MAG showed little variation indicating that the conservation of the molecular structure could be important for the function of MAG in glia axon interactions.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.