Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involvingcGMP and Ca2+ as second messengers

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane fractions. Agents that increase intracellularcAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system bycGMP. In fact, permeant analogs ofcGMP, dibutyrylcGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition ofcGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca²⁺ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca²⁺ ions.     

Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.     

The mite Varroa jacobsoni does not transmit American foulbrood from infected to healthy colonies

The present study was conducted to determine whether Varroa jacobsoni can transmit American foulbrood (AFB), caused by the bacterium Paenibacillus larvae to healthy colonies by the surface transport of spores. Five two-storey Langstroth colonies of Apis mellifera ligustica were infested by placing a sealed brood comb, with 10% Varroa prevalence, between the central brood combs of each colony. Two months later the colonies were inoculated with P. larvae by adding brood comb pieces with clinical signs of AFB (45±5 scales per colony). After 60 days the brood area was completely uncapped by means of dissecting needles and tweezers, separating the Varroa mites from the larvae and the collected mites were introduced at a rate of 51 per colony into four recipient hives placed in an isolated apiary. Twenty female Varroa specimens were separated at random and observed by SEM. Paenibacillus larvae spores were found on the dorsal shield surface and on idiosomal setae. All colonies died after 4–5 months due to a high incidence of varroosis. No clinical AFB symptoms or P. larvae spores were observed in microscopic preparations. It is concluded that Varroa jacobsoni does not transmit AFB from infected to healthy colonies; it does, however transport P. larvae spores on its surface.     

Relative biological effectiveness measurements using murine lethality and survival of intestinal and hematopoietic stem cells after fermilab neutrons compared to JANUS reactor neutrons and 60Co gamma rays

The relative biological effectiveness (RBE) of the 25-MeV (average energy) neutron beam at the Fermi National Accelerator Laboratory was measured using murine bone marrow (LD50/30) and gut (LD50/6) lethality and killing of hematopoietic colony forming units (CFU-S) or intestinal clonogenic cells (ICC). The reference radiation was 60Co gamma rays. The LD50/30 and LD50/6 for mice exposed to the Fermilab neutron beam were 6.6 and 8.7 Gy, respectively, intermediate between those of JANUS neutrons and 60Co gamma rays. The D0 values for CFU-S and ICC were 47 cGy and 1.05 Gy, respectively, also intermediate between the lowest values found for JANUS neutrons and the highest values found after 60Co gamma rays. The split-dose survival ratios for CFU-S at intervals of 1-6 hr between doses were essentially 1.0 for both neutron sources, while the corresponding split-dose survival ratio for 60Co gamma rays was consistantly above 1, reaching a maximum of 1.7 with a 1-hr interval between doses. The 3-hr split-dose survival ratios for ICC were 1.0 for JANUS neutrons, 1.85 for Fermilab neutrons, and 6.5 for 60Co gamma rays. The RBE estimates for LD50/30 were 1.5 and 2.3 for Fermilab and JANUS neutrons, respectively. Based on LD50/6, the RBEs were 1.9 (Fermilab) and 3.0 (JANUS). The RBEs for CFU-S D0 were 1.4 (Fermilab) and 1.9 (JANUS) and for jejunal microcolony D0 1.4 (Fermilab) and 2.8 (JANUS).     

Incorporation of [14C]cinnamate into hydrolase-resistant components of the primary cell wall of spinach

[¹⁴C]Cinnamate was taken up very rapidly by cultured spinach cells and completely incorporated into low-MW conjugates within 20 min. The ¹⁴C-labelled products were similar whether the [¹⁴C]cinnamate was supplied continuously over a period of hours via a peristaltic pump or instantaneously. Radioactivity was slowly recruited from the low-MW pool into aromatic components of the cell-wall fraction. Saponification of the radioactive wall fraction yielded, in addition to radioactive ferulate and p-coumarate, large amounts of ethyl acetate-soluble radioactive material with the properties of oxidatively coupled phenols. The coupled material was associated with the most highly ‘Driselase’-resistant fractions of the cell wall. In contrast, ‘Driselase’ released most of the wall's ferulate and p-coumarate on disaccharide fragments. It is suggested that the oxidatively coupled phenols are formed from simpler phenols by peroxidase and that they cross-link the polysaccharides to which they are attached, making these polysaccharides relatively ‘Driselase’-resistant.     

Modulation of platelet responsiveness through selective phosphorylation of plasma membrane proteins: Antagonistic eeffects of cyclic AMP and cyclic GMP

³²P phosphorylation of plasma membranes from human blood platelets, under conditions that closely resemble physiological ones (endogeneous phosphate donors and intact platelets in homologous plasma), result in the incorporation of the label mainly in a membrane glycoprotein of apparently high molecular weight (greater than 400 000). Dibutyryl cyclic AMP, an inhibitor of platelet aggregation, specifically increases the degree of phosphorylation of this glycoprotein. Moreover, it has been found that prostaglandin E₁ one of the most potent inhibitors of platelet aggregation which also increases phosphorylation of the same glycoprotein, is significantly more effective than cyclic AMP.Cyclic GMP does not have any apparent effect on platelet aggregation. However, incubation of platelet-rich plasma with both cyclic GMP and cyclic AMP results in a partial recovery of the platelet responsiveness towards ADP-induced aggregation. Coincidently, the degree of phosphorylation of the high molecular weight glycoprotein under these conditions, although still higher than in controls (no nucleotides added), is significantly decreased as compared with cyclic AMP-treated cells. Furthermore, cyclic GMP inhibits the cyclic AMP-dependent protein kinase activity in isolated platelet plasma membranes.These results suggest a central role for this membrane phosphoglycoprotein in the triggering of platelet aggregation and, furthermore, suggest that modulation of its degree of phosphorylation may be exerted through some cyclic AMP/cyclic GMP relationship, which in the basal state might be critical for platelet responsiveness.     

Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.     

Detection and resolution of closely related satellite DNA sequences by molecular cloning.

Highly repeated satellite DNAs often consist of mixtures of DNAs with closely related repeating sequences. By cloning individual molecules we have resolved the 1.705 g/cm³ satellite DNA of Drosophila melanogaster into two distinct components: poly$\text{d}\text{A-A-G-A-G}\text{T-T-C-T-C}$ and poly$\text{d}\text{A-A-G-A-G-A-G}\text{T-T-C-T-C-T-C}$. The presence of two distinct sequences within this physically homogeneous satellite DNA had not previously been detected by standard physical, chemical, or sequencing techniques. Both cloning and direct sequence analysis suggest that the five-base-pair and seven-base-pair repeating units reside on separate molecules and are not interspersed with each other.     

Alterations of the carrier-mediated transport of an anionic solute,methotrexate, by charged liposomes in Ehrlich ascites tumor cells

Summary Interaction of positively charged liposomes with Ehrlich ascites tumor cells increases the bidirectional transmembrane fluxes of the anionic folic acid analog, methotrexate. Negative liposomes reduce methotrexate influx. Stimulation of methotrexate influx by positively charged liposomes is time and concentration dependent, requiring at least a 5-min incubation with 2.5mm phosphatidylcholine containing 20% stearylamine for maximum effect. Stimulation is not appreciably reversed by washing the cells. Similar increases are observed for influx and efflux so that there is no change in the steady-state methotrexate electrochemical-potential difference across the cell membrane. The increase in influx appears to be a stimulation of the carrier-mediated transport process for methotrexate since both control and stimulated influx are abolished by the competitive inhibitor, 5-formyltetrahydrofolate or the sulfhydryl group inhibitor,p-chloromercuriphenylsulfonic acid and the Q₁₀ of the system remains unchanged. Influx of 5-methyltetrahydrofolate, which shares the same transport carrier as methotrexate, is also stimulated. However, the transport of folic acid, which is structurally similar to methotrexate but does not utilize the carrier, is unaffected. The kinetic change induced by positively charged liposomes is an increase in theV _ma ⁱⁿ , while theK _t ⁱⁿ remains unchanged. Trans-stimulation of methotrexate influx by 5-formyltetrahydrofolate occurs to the same extent in the presence or absence of positively charged liposomes. The liposomes have no apparent effect on the intracellular water, the extracellular space, or the chloride distribution ratio. The data suggest that interaction of positively charged liposomes with Ehrlich ascites tumor cells accelerates the rate of transposition of the membrane carrier system for methotrexate, altering the kinetics of transport without a change in transport thermodynamics.     

Similarity between 5'- and 3'-terminal nucleotide sequences and double-stranded RNA-derived sequences of eukaryotic mRNA

It has been reported recently that parts of the nucleotide sequences present in the 5′- and 3′-terminal regions of cytoplasmic mRNA are derived from double-stranded hairpin structures of heterogeneous nuclear RNA—a putative mRNA precursor (Naora, 1979). In order to explore the nature of double-stranded hairpin structures, using the sequencing data of human and rabbit globin mRNA and hen ovalbumin mRNA, we examined the following possibility: that certain regions of both the 5′- and 3′-terminal nucleotide sequences of mature mRNA were present in double-stranded hairpin structures covalently linked to both sides of the message sequence in the precursor mRNA molecule and that these double-stranded hairpin structures are similar to each other. The results support the above possibility by showing substantial similarity of nucleotide sequences between the 5′- and 3′-terminal regions of these mRNAs in terms of the formation of similar double-stranded hairpin structures.