Colocalization of N-CAM and N-cadherin in avian skeletal myoblasts

The cell-cell adhesion molecules, N-CAM and N-cadherin, have been shown previously to mediate myoblast interaction during cell fusion accompanying skeletal myogenesis. To study the localization of both molecules in fusion-competent myoblasts, we used antigen-specific primary antibodies and a double-labeling preembedding immuno-electron microscopy technique. Ultrastructural observations and quantitative analysis of the results reveal that N-CAM and N-cadherin frequently colocalize in clusters on the myoblast plasma membrane. The data provide morphological evidence that the two adhesion glycoproteins cooperate in mediating myoblast interaction during myoblast fusion.     

Absence of limbs and gross body wall defects: an epidemiological study of related rare malformation conditions.

The study is based on almost 10 million births and reports on 215 infants with two unusual malformations: amelia and gross body wall defect. Amelia without body wall defect was present in 116 cases, 67 had body wall defects without amelia, and 32 had both. The total rate was 2.2 per 100,000 births. The infants were divided into five mutually exclusive groups. There were 40 infants (0.4 per 100,000) with agenesis of the body stalk, 18 with amelia and other types of gross body wall defects (0.2 per 100,000), 56 with amelia and malformations other than gross body wall defects (0.6 per 100,000), 41 with amelia (with or without other limb reduction defects) but no nonlimb malformations (0.4 per 100,000), and 60 infants with gross body wall defects of a type other than agenesis of body stalk and without amelia (0.6 per 100,000). A weak trend of decreasing prevalence of these malformations was found during the observation period. Infants with agenesis of the body stalk and infants with amelia combined with other types of gross body wall defects occurred at an increased rate in infants of young women. This maternal age effect is also found with gastroschisis, but not with omphalocele, and may indicate etiological or pathogenetic similarities between gastroschisis and the two former groups of defect. In infants with amelia, additional limb reduction defects could be of any type: transverse, longitudinal, or intercalary. Therefore, amelia may be the end result of different types of disturbances of limb morphogenesis. There was an increased rate of twinning. The relationship with amniotic band syndrome is discussed.     

Development of efficient suicide mechanisms for biological containment of bacteria.

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.     

The amino acid sequence of a major protein component in the light harvesting complex of the green photosynthetic bacterium Chlorobium limicola f. thiosulfatophilum

A 7.5-kDa protein has been isolated from chlorosomes of Chlorobium limicola f. thiosulfatophilum and the complete primary structure determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The 74-residue protein shows great homology to a similar protein of unknown function which has been isolated from Pelodictyon luteolum but otherwise no significant homology to other proteins can be found. The possible role of the protein in the structure and function of the chlorosome is discussed.     

Immunohistochemical identification of tubular segments in percutaneous renal biopsies

Summary To identify the renal cortical tubular segments involved in tubulo-interstitial disease in formalin-fixed, paraffin-embedded percutaneous kidney biopsies, we developed multiple immunolabeling protocols using segment-specific tubular markers. The present study of biopsies from patients with minimal change or thin basement membrane nephropathy provides a baseline for interpretation of histopathology. Proximal tubules were stained either by the PAS reaction or by the biotinylated Phaseolus vulgaris erythroagglutinin (PHA-E)-streptavidin-gold-silver system (brush borders black). The anti-Tamm-Horsfall (THP) antibody-immunoperoxidase (aminoethylcarbazole, AEC-IPO), and anti-epidermal cytokeratins (ECK) antibodies-immunoalkaline-Fast Blue BB methods marked the distal straight tubules and the cortical collecting system red-brown and blue, respectively. When these immunolabelings were combined, the coapplication of AEC-PO-labeled peanut agglutinin (PNA) or anti-epithelial membrane antigen antibody-AEC-IPO technique (both are markers for distal nephron) visualized the apical membranes of distal convoluted tubules. In the protocol PHA-E + PNA + THP + ECK, the tubular basement membranes were outlined by the anti-laminin antibody-AEC-IPO staining, carried out simultaneously. The protocol PNA + THP + ECK + PAS was found to be a quite appropriate multiple immunolabeling method for the tubules, and is recommended for use as a tool in the study of tubulo-interstitial diseases.Abbreviations PAS periodic acid-Schiff reaction - PHAE Phaseolus vulgaris erythroagglutinin - PNA Peanut agglutinin - EMA epithelial membrane antigen - THP Tamm-Horsfall glycoprotein - ECK epidermal cytokeratins - PO peroxidase - Biot-PHA-E biotinylated PHA-E - APAAP complexes of alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase - SWARI swine anti-rabbit immunoglobulins - FCS fetal calf serum - TBS Tris-buffered saline - AEC aminoethylcarbazole - DAB diaminobenzidine - FBBB Fast Blue BB - IA immunoalkaline - GL glomerulus - PT proximal tubule - DST distal straight tubule - DCT distal convoluted tubule - CCS cortical collecting system - CT connecting tubule - CD collecting duct     

Levels of hypoxanthine phosphoribosyltransferase RNA in human cells

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other “housekeeping genes,” thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3–0.8 pg/μg DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8–1.2 pg/μg DNA, which corresponds to 6–10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5–10 h before the onset of DNA synthesis, and a steady-state level was reached after 2–3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (LeschNyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.     

Reversal by EGTA of the enhanced secretory responsiveness of mast cells due to treatment with ouabain

The effect of EGTA on the enhancement by ouabain of compound 48/80-induced secretion from mast cells was compared with the effect on the Na(+)-K+ pump activity. The time-dependent secretory enhancement by ouabain was blocked by addition of EGTA to the cell suspension concomitantly with the addition of ouabain, and EGTA caused a large increase in the pump activity. Addition of 10 microM EGTA to ouabain-treated cells stopped but did not reverse the enhancement. The experiments show that the effect of ouabain was due to changes in a calcium pool utilized in compound 48/80-induced secretion following changes in the Na+,K+ pump activity.     

A role for the neural cell adhesion molecule, NCAM, in myoblast interaction during myogenesis

The Ca2(+)-independent neural cell adhesion molecule, NCAM, is expressed by both nerve and muscle cells and has been shown to mediate both nerve-nerve and nerve-muscle cell interaction. A role for NCAM in muscle-muscle cell interaction has been proposed but not demonstrated. Here we report evidence that NCAM is expressed by embryonic chick muscle cells during in vitro development and functions together with Ca2(+)-dependent adhesion molecules in mediating myoblast interaction during the formation of multinucleate cells.     

Continuous flow microcalorimetric measurement of heat production in white adipose tissue from obese (ob/ob) mice and their lean littermates

Heat production, free fatty acid and glycerol release from white adipose tissue fat pads from obese (ob/ob) mice and their lean littermates are determined. Heat production was significantly lower in obese mice compared to lean mice when expressed on wet weight basis but not when expressed on DNA basis. Noradrenaline significantly increased the heat production in fat pads from both groups of animals. However, the increase in heat production due to noradrenaline addition in fat pads from lean mice was significantly higher than in fat pads from obese mice. The release of free fatty acids and glycerol before incubation with noradrenaline was similar from fat pads from both groups of animals. Addition of noradrenaline to the fat pads increased the release of free fatty acids and glycerol in both groups of animals, but the increase was significantly larger from fat pads from lean mice. In the absence of noradrenaline the free fatty acid/glycerol ratio (mol/mol) in the effluent was 7.9:1 and 4.8:1 for lean mice and obese mice, respectively. In the presence of noradrenaline the ratio decreased to 3:1 for both groups of animals.