Ultrastructural studies of initial stages of mineralization of long bones and vertebrae in human fetuses

We studied 27 embryos of 5-12 weeks gestational age where pregnancy was interrupted due to paramedical reasons, in order to find the developmental stages at which matrix vesicles appear in cartilage, and whether they are involved in the mineralization process. Specimens of long bones, lumbar and thoracic vertebral column were prepared for light, transmission and scanning electron microscopic studies. In the cartilaginous models of long bones, matrix vesicles were found amongst maturing and hypertrophic chondrocytes already by the 6th week after fertilization. By that stage, bone rudiments consisted of only cartilage that was not yet mineralized. In the vertebral column matrix, vesicles were found in the vertebral bodies amongst maturing and hypertrophic chondrocytes at the beginning of the 8th week. At that stage, although hypertrophy of chondrocytes was observed, mineralization was still absent. No matrix vesicles were found in the perichondrium, investing mesenchyme and intervertebral discs. Mineralization of cartilage in long bone rudiments started in the form of hydroxyapatite crystals within or around the matrix vesicles at 7 weeks of age and in the vertebral column at 11 weeks. As mineralization progressed, more hydroxyapatite crystals were observed around the matrix vesicles, forming typical calcospherites . Mineralization then progressed in the form described in other animals.     

Cloning of the natural gene for the sweet-tasting plant protein thaumatin

Five different clones, homologous to the structural gene for the sweet-tasting plant protein thaumatin, have been isolated from leaf DNA of Thaumatococcus daniellii Benth. Restriction maps, hybridization studies, S1-nuclease mapping and R-loop formation revealed that the thaumatin genes isolated belong to one multigene family, and have two very small introns situated at different positions in the various structural genes. A similar situation prevails in a number of seed storage genes. This suggests a similarity between the sweet-tasting protein thaumatin and seed storage proteins.     

Catecholamines in fetal pig plasma and the response to acute hypoxia and chronic fetal decapitation

Summary Dopamine, norepinephrine and epinephrine were measured by radioenzymatic assay in blood plasma samples drawn from the umbilical arteries of 30 anaesthetised Landrace pig fetuses. Just prior to term, the concentrations of dopamine (0.46±0.14 ng·ml^–1) and norepinephrine (1.74±0.60 ng·mg^–1) were lower than earlier in gestation, whereas epinephrine concentrations at term (0.80±0.31 ng·ml^–1) were similar to those at mid-gestation, intervening stages of gestation having higher levels of plasma epinephrine. Fetal hypoxia was induced by clamping the umbilical cord for 2 min and the catecholamines determined in arterial blood samples immediately thereafter, then again 3 min after removal of the clamp. Inconsistent effects of cord clamping on catecholamine levels were seen at 55 days, but thereafter, in all but one instance, the hormone levels were increased. Fetuses near term tended to respond less than fetuses at 75 and 96 days gestation (term=114±1 day). Catecholamines were also present in the circulation of fetuses decapitated at 42 days gestation and studied at 109±1 days. The average concentrations of dopamine (1.12±0.27 ng·ml^–1) and norepinephrine (8.23±3.04 ng·ml^–1) were greater than in intact fetuses, the plasma epinephrine levels being comparable to, or slightly higher than, those in intact fetuses. The results demonstrate that catecholamines are present in the circulation of the intact and decapitated pig fetus and that the actual concentrations and the type of response to umbilical cord clamping are dependent on gestation age.     

Architecture of the outer membrane of Escherichia coli K12. II. Freeze fractur morphology of wild type and mutant strains

Freeze fracturing electron microscopy of Escherichia coli K12 cells showed that the outer fracture face of the outer membrane is densily occupied with particles. On the inner fracture face of the outer membrane, pits are visible, which are probably complementary to the particles at opposite fracture face. This observation suggests that the particles are micelle-like. In some mutants which lack one or more major outer membrane proteins the density of particles is reduced. The loss of protein d appeared to a prerequisite for this phenomenon. However, mutants which lack all glucose and heptose-bound phosphate in their lipopolysaccharide also have a reduction in particle density whereas, the amount of protein d is normal. Moreover, loss of lipopolysaccharide by EDTA treatment also caused a reduction in the density of particles. From these results it is hypothesized that the particles consist of lipopolysaccharide aggregates stabilized by divalent cations and probably complexed with protein and/or phospholipid.     

Retinoic acid induces transglutaminase activity but inhibits cornification of cultured epidermal cells

In cultured mouse epidermal basal cells, retinoic acid is a potent inducer of transglutaminase, the enzyme responsible for isodipeptide bond formation in protein cross-linking in the production of the cornified membrane during terminal differentiation. Paradoxically retinoic acid also inhibits the formation of the cross-linked envelope and greatly reduces the level of dipeptide bond formation in epidermal cells induced to differentiate by calcium. These results suggest a novel mechanism by which retinoids can modify transglutaminase activity and epidermal differentiation.     

meoA is the structural gene for outer membrane protein c of Escherichia coli K12

Summary The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Mel resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptorcomplex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c^*. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.     

Architecture of the Outer Membrane of Escherichia coli III. Protein-Lipopolysaccharide Complexes in Intramembraneous Particles

In a previous paper (A. Verkleij, L. van Alphen, J. Bijvelt, and B. Lugtenberg, Biochim. Biophys. Acta 466:269-282, 1977) we have hypothesized that particles on the outer fracture face of the outer membrane ([Formula: see text]), with corresponding pits on the inner fracture face of the outer membrane ([Formula: see text]), consist of lipopolysaccharide (LPS) aggregates stabilized by divalent cations and that they might contain protein and/or phospholipid. In the present paper the roles of LPS, cations, and proteins in these [Formula: see text] particles are described more extensively, using a strain that lacks the major outer membrane proteins, b, c, and d (b(-) c(-) d(-)), and has a reduction in the number of [Formula: see text] particles of 75%. To study the role of divalent cations in the formation of [Formula: see text] particles, these b(-) c(-) d(-) cells were grown or incubated with Ca(2+), Mg(2+), or putrescine. The presence of Ca(2+) resulted in the appearance of many [Formula: see text] particles and [Formula: see text] pits. Mg(2+) and putrescine were less effective than Ca(2+). Introduction of these particles was not accompanied by alterations in the relative amounts of LPS and cell envelope proteins. Ca(2+) treatment of a heptoseless derivative of a b(-) c(-) d(-) strain did not result in morphological changes. Incubation of Ca(2+)-treated cells with ethylenediaminetetraacetate caused the disappearance of the introduced particles as well as the release of more than 60% of the cellular LPS. These results strongly support the hypothesis that LPS is involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The roles of various outer membrane proteins in the formation of [Formula: see text] particles were studied by comparing the freeze-fracture morphology of b(-) c(-) d(-) cells with that of cells which contain one of the outer membrane proteins b, c, d, and e or the receptor protein for bacteriophage lambda. The results showed that the presence of any of these five proteins in a b(-) c(-) d(-) background resulted in a large increase in the number of [Formula: see text] particles and [Formula: see text] pits, indicating that these proteins are, independent of each other, involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The simplest explanation for the results is that in wild-type cells each particle consists of LPS complexed with some molecules of a single protein species, stabilized by either divalent cations or polyamines. It is hypothesized that the outer membrane of the wild-type cell contains a heterogeneous population of particles, of which 75% consists of protein b-LPS, protein c-LPS, and protein d-LPS particles. A function of these particles as aqueous pores is proposed.     

The two-step interaction between α-dimethylaminonaphthalene-1-sulfonyl-pepsinogen-(1–12) and pepsin

The binding between α-dimethylaminonaphthalenesulfonyl-(1–12) and porcine pepsin can be detected by the large changes that occur in the fluorescence spectra of the dimethylaminonaphthalenesulfonyl chromophore due to energy transfer from tryptophan residues of the protein. The interaction was previously shown to consist of two steps: a fast step leading to a greatly enhanced fluorescence followed by a slower rearrangement step which reduces the fluorescence but leads to tighter binding and inhibition of the catalytic activity of pepsin (1). The two steps have been studied over a wide range of values of pH, temperature, and ionic strength to gain additional insights into the physical events occurring during the interaction. Based on the pH and ionic strength dependence, the initial step most likely involves electrostatic interaction of the basic peptide inhibitor with the acidic surface of pepsin in a rapid collision process. The use of this fluorescent reporter group has also suggested that the equilibrium binding after the slower rearrangement may also be pH dependent with most effective binding at higher pH. The kinetics of the slow step were measured by monitoring the continuous fluorescence decay. The resulting rates are compared to the rates observed by others for binding of pepstatin to pepsin. From the pH dependence of fluorescence, pK_app values are obtained for the dansylated peptide (3.25), for the pH dependence of the initial binding step (4.87), and for the equilibrium position (4.75).     

The recombination activating genes,RAG 1 and RAG 2, are on chromosome 11p in humans and chromosome 2p in mice

The recombination activating genes RAG-1 and RAG-2 are adjacent genes that act synergistically to activate variable-diversity-joining (V(D)J) recombination. Southern analysis of hybrid cell lines derived from patients with the Wilms tumor-aniridia-genitourinary defects-mental retardation (WAGR) syndrome and from mutagenized cell hybrids selected for deletions in chromosome 11 has allowed us to map the chromosomal location of the human RAG locus. The RAG locus defines a new interval of human chromosome 11p, but is not associated with any genetically mapped human disease. Guided by the chromosomal localization of the human recombination activating genes, we have also mapped the location of the mouse Rag locus.