A sensitive method to introduce membrane-bound proteins into recipient cells based on affinity enrichment of lipid vesicles to the recipient cell prior to fusion

A sensitive analytical procedure for studying membrane-bound structures has been developed. Membrane glycoproteins inserted into liposomes were transferred to recipient cells by use of a lectin, concanavalin A, bound to the cells as a bridge to generate proximity between the recipient cell and the glycoprotein-containing liposome, prior to exposure to the fusing agent, poly(ethylene glycol). Partially purified histocompatibility antigen from rats was introduced into the membrane of human lymphocytes. After treating the cells with poly(ethylene glycol) under fusion conditions, some of the antigen present in the preparation could not be eluted with alpha-methyl mannoside and EDTA, indicating that incorporation in the cell membrane had taken place. This antigen remained exposed on the lymphocyte surface for approximately 1 h as demonstrated by sensitivity of the lymphocytes to the lytic effect of an antiserum to the histocompatibility antigen in the presence of complement. Some of the lectin molecules seemed to be internalized in the cells but no induction of cell mitosis was observed. The described method gives an opportunity to work with small amounts of membrane proteins inserted into liposomes, introducing them into recipient cells for analysis of their biological activities.     

Connections between the cristae and the surface of rat heart muscle mitochondria

Contrary to the generally accepted rule that there are only two fracture faces associated with a membrane, the analysis of double replicas at rat heart muscle mitochondria revealed three pairs of complementary replicas with one face in each pair exposing the outer surface membrane. The replicas must then expose the surfaces of the outer surface membrane and in two of the pairs the fracture had passed between the two surface membranes in two alternative ways, either clearly between the two membranes or the fracture deviated into and through the inner surface membrane at regularly spaced intervals. This deviation reveals that at these sites the connection between the two surface membranes is particularly firm. The analysis led to the conclusion that these sites correspond to those where the stalk-like connections extending from the cristae are connected to the inner surface membrane. This way proteinaceous pathways connect the cristae to the surface of the mitochondria.     

Histochemistry and ultrastructure of adrenergic and acetylcholinesterase-containing nerves supplying follicles and endocrine cells in the guinea-pig ovary

Summary With the use of an anti-human S-100 protein antibody, it was possible to reveal a characteristic cell type in the anterior lobe of the normal human pituitary. These cells, so-called folliculo-stellate cells, were present in all pituitaries studied but their number varied from one gland to another. Immunoreactive cells, isolated or grouped, were arranged close to various secretory granulated cells. Especially by use of double immunoenzymatic labeling, it was evident that these cells are spatially related either to somatotropes, prolactin cells and corticotropes, or to glycoprotein-containing cells. Such immunoreactive cells were rare or absent in pseudo-follicular arrangements of secretory granulated cells. Since it is now possible to identify this cell type by light microscopy and since no reliable functional significance is known, it seems more advisable to term this cell type stellate cell instead of folliculostellate cell.     

Quantitation of glucagon by radioreceptorassay

Receptors for glucagon on rat liver membranes were characterized. They bound [125I] glucagon rapidly in a specific and saturable way. Addition of unlabelled glucagon displaced [125I] glucagon from the binding sites in a concentration dependent way. Concentrations from 10(-9) to 10(-8) M of glucagon caused a linear reduction of binding of labelled glucagon. This concentration interval was used for a three-point assay which fulfilled statistical requirements for validity. Individual assays normally resulted in potency estimates of high precision and statistical weight. Mean values for glucagon activity of preparations tested by receptor assay were within the fiducial limits (P = 0.95) for corresponding activity determined by the rabbit blood glucose method. The receptor assay is less time consuming and requires only part of one rat liver while the in vivo assay uses 16 rabbits. Thus, the receptor assay is less resource demanding and should serve well as a screening instrument for control of potency of glucagon preparations.     

Animal community structure as a function of stream size

The species-area relationship of the island biogeography theory was calculated for macroinvertebrates in 22 coastal, adjacent streams. A z-value of 0.19 was obtained. The low z-value was probably a consequence of the short distances between streams as well as high dispersal rates. In addition, a cluster analysis based on the dissimilarity of species assemblages showed that stream size was of prime importance in categorizing the streams. To a smaller extent water quality affected the community structure in the streams.     

Lactate dehydrogenase in developing rat oral epithelium

Freeze-dried sagittal, whole-body sections of 10-day-old rats were incubated for lactic dehydrogenase (LDH) using different media in the presence of the inhibitors urea and fluoropyruvate. Phenazine methosulfate (PMS) and menadione, which are regularly used in current histochemical media and are believed to promote the demonstration of LDH activity, were also added and shown to be insufficient for the demonstration of total LDH activity, and PMS even seemed to have an inhibitory effect on LDH activity in oral epithelium. However, cumulated data from the different incubations show that the oral epithelium of developing rats may contain two different types of LDH, one in the basal cells with possibly aerobic characteristics, and another in the spinosum/granulosum cells with anaerobic characteristics.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.     

The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located.     

Fluid balance in exercise dehydration and rehydration with different glucose-electrolyte drinks

After exercise dehydration (3% of body weight) the restoration of water and electrolyte balance was followed in 6 male subjects. During a 2 h rest period after exercise, a drink of one of four solutions was given as 9 X 300 ml portions at 15 min intervals: control (C-drink), high potassium (K-drink), high sodium (Na-drink) or high sugar (S-drink). An exercise test (submaximal and supramaximal work) was performed before dehydration and after rehydration. Dehydration reduced plasma volume by 16%, a process reversed on resting even before fluid ingestion began, due to release of water accumulated in the muscles during exercise. After 2 h rehydration, plasma volume was above the initial resting value with all 4 drinks. The final plasma volumes after the Na-drink (+14%) and C-drink (+9%) were significantly higher than after the K- and S-drinks. The Na-drink favoured filling of the extracellular compartment, whereas the K- and S-drinks favoured intracellular rehydration. In spite of the higher than normal plasma volume after rehydration, mean heart rate during the submaximal test was 10 bpm higher after rest and rehydration than in the initial test, and was not different between the drinks. The amount of work which could be performed in the supramaximal test (105% VO2max) was 20% less after exercise dehydration and subsequent rest and rehydration than before. This reduction was similar for all drinks, and may be due to a decreased muscle glycogen content (70% of initial) at the time of the second test.