Nucleolar necklace formation in response to hemolymph ecdysteroid peaks.

Previous work on the last (fifth) larval stadium of Calpodes showed two phases of elaboration of epidermal nucleoli correlated with RNA synthesis, the first after ecdysis at the beginning of the intermolt and the second near the end of the stadium prior to molting. Both phases followed periods of elevated hemolymph ecdysteroid. The demonstration of four hemolymph ecdysteroid peaks and an improvement in the bismuth-staining procedure for nucleoli has prompted further study of nucleolar changes in relation to hemolymph edcysteroids. We have found that three of the four ecdysteroid peaks (I, II and IV) are followed by nucleolar changes. The exception is the commitment peak (III) for which there is no corresponding nucleolar change. The three nucleolar cycles are similar in their essential features. An intercycle nucleolus consists of one or a few irregularly shaped particles that become more densely stained and condense into a knot at the beginning of each cycle. The knot unfolds into a necklace which beomes beaded as it elongates to a length of about 23 mum. Cells have one or two, rarely more, necklaces presumably depending on their ploidy. At the end of the cycle the necklaces contract, becoming coarser and fragmented before they condense to the intercycle condition of central irregular cores. Whereas nucleolar necklaces are a general response to hemolymph ecdysteroids, mitoses are locally determined and are imposed over other nuclear activities at any time in the third nucleolar cycle.     

Adhesivity and rigidity of erythrocyte membrane in relation to wheat germ agglutinin binding

Binding of the plant lectin wheat germ agglutinin (WGA) to erythrocyte membranes causes membrane rigidification. One of our objectives has been to directly measure the effects of WGA binding on membrane rigidity and to relate rigidification to the kinetics and levels of WGA binding. Our other objective has been to measure the strength of adhesion and mechanics of cell separation for erythrocytes bound together by WGA. The erythrocyte membrane rigidity was measured on single cells by micropipette aspiration. The slope of the suction pressure-length data for entry into the pipette provided the measure of the membrane extensional modulus. Data were collected for cells equilibrated with WGA solutions in the range of concentrations of 0.01- 10 micrograms/ml. Erythrocyte-erythrocyte adherence properties were studied by micropipette separation of two-cell aggregates. First, a "test" cell was selected from a WGA solution by aspiration into a small micropipette, then transferred to a separate chamber that contained erythrocytes in WGA-free buffer. Here, a second cell was aspirated with another pipette and maneuvered into close proximity of the test cell surface, and adhesive contact was produced. The flaccid cell was separated from the test cell surface in steps at which the force of attachment was derived from the pipette suction pressure and cell geometry. In addition, we measured the time-dependent binding and release of fluorescently labeled WGA to single erythrocytes with a laser microfluorometry system. The results showed that the stiffening of the erythrocyte membrane and binding of fluorescently labeled WGA to the membrane surface followed the same concentration and time dependencies. The threshold concentration for membrane stiffening was at approximately 0.1 microgram/ml where the time course to reach equilibrium was close to 1 h. The maximal stiffening (almost 30-fold over the normal membrane elastic modulus) occurred in concentrations greater than 2 micrograms/ml where the time to reach equilibrium took less than 1 min. The WGA binding also altered the normal elastic membrane behavior into an inelastic, plastic-like response which indicated that mechanical extension of the membrane caused an increase in cross-linking within the surface plane. Similar to the stiffening effect, we observed that the membrane adhesivity of cells equilibrated with WGA solutions greatly increased with concentration greater than 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation, characterization and metal-ion-binding properties of the alpha-subunit from S-100a protein.

The present experiments were undertaken to investigate the role of the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) in the alpha 1-adrenergic stimulation of respiration in isolated hamster brown adipocytes. Exposure of isolated brown adipocytes to the alpha-adrenergic-receptor agonist phenylephrine provoked a breakdown of 30-50% of the PtdIns-4-P and PtdIns-4,5-P2 after prelabelling of the cells with [32P]Pi. Coincident with the breakdown of phosphoinositides was an accumulation of labelled phosphatidic acid, which continued for the duration of the cell incubation. The time course of phosphoinositide breakdown was defined more precisely by pulse-chase experiments. Under these conditions, phenylephrine caused radioactivity in phosphatidylinositol, PtdIns-4-P and PtdIns-4,5-P2 to fall by more than 50% within 30 s and to remain at the depressed value for the duration of the incubation (10 min). This phospholipid response to alpha-adrenergic stimulation was blocked by exposure of the cells to phorbol 12-myristate 13-acetate (PMA); likewise phenylephrine stimulation of respiration was prevented by PMA. beta-Adrenergic stimulation of respiration and inhibition of respiration by 2-chloroadenosine and insulin were, however, unaffected by treatment with PMA. On the assumption that PMA is acting in these cells as an activator of protein kinase C, these results suggest the selective interruption of alpha-adrenergic actions in brown adipocytes by activated protein kinase C. These findings suggest that breakdown of phosphoinositides is an early event in alpha-adrenergic stimulation of brown adipocytes which may be important for the subsequent stimulation of respiration. The results from the pulse-chase studies also suggest, however, that phenylephrine-stimulated breakdown of inositol phospholipids is a short-lived event which does not appear to persist for the entire period of exposure to the alpha 1-adrenergic ligand.     

Modification of susceptibility to Klebsiella pneumoniae during murine cytomegalovirus infection

The effect of murine cytomegalovirus (MCMV) infection on susceptibility to bacterial infection was studied in mice by a combination of intraperitoneal (ip) inoculation of a sublethal dose of MCMV with subsequent ip challenge of 2 X 10(3) cfu of a strain of Klebsiella pneumoniae (KP). When given alone, KP produced a mortality of 30-40%. Mortality was increased when KP was given 1 to 7 days after MCMV injection with the peak increase at the 4th to 5th day when 100% mortality occurred. Virus levels in various organs of mice infected with MCMV alone, or superinfected with KP did not differ. Bacterial counts on the other hand, showed that increased mortality in mixed MCMV and KP infected mice was due to an uncontrolled growth of bacteria at the site of primary lodgment, i.e., the peritoneum, and severe systemic infection. Neutrophil response to growth of KP during the first 3 days of bacterial infection was defective in MCMV infected mice. While the initial clearance of KP from the blood was more efficient in MCMV infected mice, probably due to activated reticuloendothelial function, it did not protect the mice against KP infection. Using the in vivo model, it was shown that poor neutrophil response and possibly other defective neutrophil functions were responsible for increased mortality to KP infection in MCMV infected mice.     

Long-acting contraceptive agents: Norethisterone esters of arylcarboxylic acids

The synthesis of esters of norethisterone (17α-ethynyl-17β-hydroxy-estr-4-en-3-one) with acids containing a benzene ring is described, two methods of esterification being compared in terms of yield and convenience. The activities of these esters as long-acting contraceptive agents have been evaluated.     

Purification and Properties of 4-Hydroxy-2-Ketopimelate Aldolase from Acinetobacter

The chemical synthesis of 4-hydroxy-2-ketopimelic acid is described. An aldolase that cleaves this compound to succinic semialdehyde and pyruvate has been purified from Acinetobacter grown at the expense of 4-hydroxyphenylacetic acid. The molecular weight of the enzyme was about 158,000 from sedimentation equilibrium data; other physical determinations gave values in reasonable agreement. The protein was globular and was dissociated in sodium dodecyl sulfate to give a species of molecular weight 25,700. The enzyme attacked both enantiomers of synthetic 4-hydroxy-2-ketopimelate and was stimulated by Mg(2+) and Mn(2+) ions.     

Molecular architecture of adult locust cuticle at the electron microscope level

It is shown that locust adult endocuticle consists of a daily alternation of two types of chitin-protein architecture: (i) non-lamellate day layers with microfibrils oriented in a preferred direction, traversed by pore canals whose shape resembles an untwisted ribbon, (ii) lamellate night layers with helicoidally oriented microfibrils traversed by pore canals shaped like regularly twisted ribbons. Uncoupling the circadian clock which normally controls the timing of these two types leads to growth of cuticles which are organized like one or the other throughout. We can thus experimentally change the architecture of the microfibrils which in turn changes the pore canals.     

Spider toxins selectively block calcium currents in Drosophila

Toxins from spider venom, originally purified for their ability to block synaptic transmission in Drosophila, are potent and specific blockers of Ca2+ currents measured in cultured embryonic Drosophila neurons using the whole-cell, patch-clamp technique. Differential actions of toxins from two species of spiders indicate that different types of Drosophila neuronal Ca2+ currents can be pharmacologically distinguished. Hololena toxin preferentially blocks a non-inactivating component of the current, whereas Plectreurys toxin blocks both inactivating and non-inactivating components. These results suggest that block of a non-inactivating Ca2+ current is sufficient to block neurotransmitter release at Drosophila neuromuscular junction.