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.     

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

Single-file diffusion through the Ca2+-activated K+ channel of human red cells

Summary We have examined the effect of internal and external pH on Na⁺ transport across toad bladder membrane vesicles. Vesicles prepared and assayed with a recently modified procedure (Garty & Asher, 1985) exhibit large, rheogenic, amiloridesensitive fluxes. Of the total²²Na uptake measured 0.5–2.0 min after introducing tracer, 80±4% (mean±se,n=9) is blocked by the diuretic with aK ₁ of 2×10^–8 m. Thus, this amiloridesensitive flux is mediated by the apical sodium-selective channels. Varying the internal (cytosolic) pH over the physiologic range 7.0–8.0 had no effect on sodium transport; this result suggests that variation of intracellular pHin vivo has no direct apical effect on modulating sodium uptake. On the other hand,²²Na was directly and monotonically dependent on external pH. External acidification also reduced the amiloride-sensitive efflux across the walls of the vesicles. This inhibition of²²Na efflux was noted at external Na⁺ concentrations of both 0.2 m and 53mm.These results are different from those reported with whole toad bladder. A number of possible bases for these differences are considered and discussed. We suggest that the natriferic response induced by mucosal acidification of whole toad urinary bladder appears to operate indirectly through one or more factors, presumably cytosolic, present in whole cells and absent from the vesicles.     

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.     

Radioimmunoassay for determination of indole-3-acetic acid in fungi and plants

A radioimmunoassay technique for indole-3-acetic acid is described. The method has successfully been used to measure extractable indole-3-acetic acid in fungal and plant materials and is able to detect as little as 0.3 pmol. As non-radioactive antigen the methyl ester of indole-3-acetic acid is used and the radioactive antigen is tritiated. An acid-catalyzed esterification of indole-3-acetic acid is used for conversion into methyl ester. The measuring range of the assay is 0.3–10 pmol. In the assay, separation of free and bound fractions is achieved by dextran-coated charcoal, leaving the bound fraction in the supernatant.     

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.     

Regional Distribution of Homocysteine in the Mammalian Brain

The regional distribution of L-homocysteine (Hcy) was determined in brains from mouse, rat, guinea pig, and rabbit, using a sensitive radioenzymatic assay. Large interspecies variations in the Hcy content in various parts of the brain were observed, but cerebellum contained the highest amount in all species investigated. In the rat the amount of Hcy in cerebellum (6.4 nmol/g) was about sixfold higher than in most other parts of the brain, whereas in the mouse and guinea pig the amount in cerebellum (about 1 nmol/g) was only twofold higher than in the other brain regions. There was a remarkably high level of Hcy in all regions of the rabbit brain (4-10 nmol/g); the highest concentration was found in the cerebellar white matter. In this species the amount of Hcy in all brain regions examined exceeded that in the liver.     

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.