Zn+2 induces reversible cross-linking of human placental thyroid hormone nuclear receptor with no effect on hormone binding.

Zn+2 is required for specific binding of c-erbA proteins to the hormone response elements of target genes. It is unclear whether Zn+2 is important for the binding of ligand to c-erbA proteins. The present study evaluated the effect of Zn+2 and other divalent cations on the binding of 3,3',5-triiodo-L-thyronine(T3) to the purified human placental c-erbA protein (h-TR beta 1). Zn+2 induced cross-linking of h-TR beta 1 to form aggregates in a dose-dependent manner with an apparent half-maximal concentration of approximately 200 microM at 22 degrees C. Cross-linking was reversible by the addition of 5 microM EDTA or 10 mM dithiothreitol. The cross-linked h-TR beta 1 bound T3. These results indicated Zn+2 had no effect on T3 binding and suggested that the cysteines and histidines involved in cross-linking are not essential for T3 binding.     

Identification of bacterial chemoattractants for oyster (Crassostrea virginica) hemocytes

The cytoplasmic and cell wall components of the Gram-positive bacterium Bacillus megaterium and the cytoplasmic and cell envelope components of the Gram-negative bacterium Escherichia coli were assayed for chemotactic activity for the hemocytes of Crassostrea virginica. The cellular components were separated by differential centrifugation and gel filtration was used to determine the approximate molecular weights of the chemoattractant molecules. Active fractions were assayed for glycoproteins and lipoproteins. As a result, it is known that hemocytes are chemotactically attracted to proteins of approximately 10,000 daltons which are associated with the cell wall of B. megaterium and the cell envelope of E. coli.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

HISTOLOGICAL ANALYSIS OF ADVENTITIOUS BUD FORMATION IN CULTURED DOUGLAS FIR COTYLEDON

Initiation of adventitious bud formation in vitro from Douglas fir cotyledons required both cytokinin and auxin at concentrations of 5 μM BAP and 5 nM NAA. Histological observations showed that these adventitious buds arose de novo from cells residing in hypodermal layers. Development of adventitious buds in culture was characterized by the sequential appearance of four anatomically distinguishable structures: 1) meristemoid, 2) bud primordium, 3) shoot apex with needle primordia, and 4) adventitious bud. The anatomical structure of tissue culture-produced buds was similar to that of vegetative buds produced on intact plants. Cultured cotyledons capable of producing adventitious buds (bud culture) were compared with bud-callus and callus cultures initiated by 5 μM BAP plus 5 μM NAA and 5μM NAA alone without BAP, respectively. Results showed that, during early stages of the culture period (i.e., prior to the appearance of meristemoid structure), cell division of bud culture was mainly located in hypodermal layers, whereas for the other culture types, bud-callus and callus cultures, cell division occurred randomly in all tissues.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle.

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Specific D-glucose transport in sarcolemma vesicles.

The sarcolemmal fraction prepared from rat skeletal muscle consists of osmotically active vesicles that accumulate D-glucose in preference to L-glucose, apparently by facilitated diffusion into intravesicular space. Stereospecific D-glucose uptake by these vesicles is a saturable rpocess, inhibited by phloridzin, by cytochalasin B, and by certain sugars, and enhanced by counterflow. An additional leak pathway permits entry of both D- and L-glucose into the vesicles. Stereospecific D-glucose transport by sarcolemmal vesicles is enhanced to a small extent by insulin, provided the hormone is administered prior to cell disruption. In membranes prepared from insulin-pretreated muscle, Ca2+ produces a small further enhancement. Local anesthetics preferentially inhibit stereospecific D-glucose transport. Apparent uptake of both D- and L-glucose is greater when vesicles are suspended in salt solutions rather than sucrose, an effect attributed to increased functional vesicular volume.     

The inhibition of sugar transport and oxidation in fat cell ghosts by colchicine.

Colchicine inhibits glucose oxidation and the uptake of 2-deoxy-D-glucose in fat cell ghosts but has no effect on glucose oxidation by fat cell homogenates. This inhibition is rapid, reversible, and temperature-independent. Insulin-stimulated glucose oxidation and 2-deoxy-D-glucose transport are also inhibited by colchicine to an extent comparable to the basal processes.     

A quantitative study of phagocytosis by hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria

Fresh hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria were exposed to known concentrations of Bacillus megaterium, Escherichia coli, and Staphylococcus aureus in vitro and it was ascertained that all four types of cells of C. virginica and all three types of M. mercenaria became associated with the bacteria. Association is defined as either the first, i.e., contact and adherence, or second, i.e., engulfment, phase of phagocytosis. However, when the surfaces of each type of cell, as well as the percentages of each type in whole hemolymph, from both species of molluscs are taken into consideration, it is concluded that the granulocytes are the most important from the standpoint of phagocytosis.When hemocytes of M. mercenaria were exposed to Bacillus megaterium at 4°, 22°, and 37°C, it was found that the association indices were higher at the latter two temperatures. It is postulated, because of the results of Feng and Feng (1974), that nonself materials adhere with less frequency at 4°C and hence are not phagocytosed at this lower temperature.     

Properties of follicle-stimulating-hormone receptor in cell membranes of bovine testis.

A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 X10(-11)M, and 5.9 X 10(-14)mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40 degrees C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5mu/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone.