The cardiac sarcoplasmic reticulum - glycogenolytic complex, an internal beta adrenergic receptor.

Agents which stimulate the formation of cyclic AMP are well known to cause positive inotropic effects on the heart. This review suggests that the myocardial cell is multicompartmented and has several effector sites for cyclic AMP-mediated events and therefore several possible receptor sites for cyclic AMP-mediated agents. It is possible that some of the complex relationships between cyclic AMP-mediated agents and cardiac physiologic functions result from differences among agonist receptors and enzymes in the intracellular compartments. Examination of the characteristics of intracellular drug receptors may yield a new frontier for cardiac pharmacology.     

Intracellular Ca2+ release by flufenamic acid and other blockers of the non-selective cation channel.

We report in this paper using measurement of intracellular free Ca2+ with fura-2, that flufenamic acid and several related blockers of the 25 pS Ca(2+)-activated non-selective cation channel cause release of Ca2+ from an intracellular store other than the endoplasmic reticulum, possibly from mitochondria. A new compound, 4'-methyl-DPC, is found to be as effective in blocking non-selective cation channels as other flufenamate analogs but, like the parent compound, the non-selective cation channel blocker DPC, it does not cause release of Ca2+ from intracellular stores. DPC and 4'-methyl-DPC are thus the most suitable of the available blockers of non-selective cation channels for use in studies on the role of these channels in normal cell function.     

Growth orientation of heart cells on nylon monofilament. Determination of the volume-to-surface area ratio and intracellular potassium concentration

A new method is described for orienting the growth of embryonic chick heart cells as thin annuli about nylon monofilament. Analytical measurements of cell water, intracellular potassium, cell volume, and cell surface area incorporate several new techniques and provide the quantitative basis for characterizing the respective cell types in the preparation. The measurements support the hypothesis that tissue culture methodology does not alter the morphological and physiological properties of cardiac muscle cells. The preparations are ideally suited for radiotracer studies since tissue mass can be increased while retaining a relatively short diffusional distance.     

Interactions between second messengers: cyclic AMP and phospholipase A2- and phospholipase C-metabolites.

The article reviews several new findings on the interactions between phospholipase A2- and phospholipase C-derived metabolites and cyclic AMP, in view of the developments recently achieved in studies on intracellular signal transduction. A complex network of multi-directional regulative mechanisms in the airways and inflammatory blood cells is briefly outlined.     

Galtierella biscalithecae nov. gen. et sp., a Late Pennsylvanian endophytic water mold (Peronosporomycetes) from France

A new fossil water mold (Peronosporomycetes), Galtierella biscalithecae nov. gen. et sp., consisting of coenocytic hyphae occurs as an intracellular endophyte in a partially degraded specimen of the reproductive organ Biscalitheca cf. musata (Zygopteridales) from the Upper Pennsylvanian Grand-Croix cherts (Saint-Étienne Basin, France). Some hyphal tips produce small spheres that subsequently develop into ornamented, opaque-walled oogonia; amphigynous antheridia encircle the necks of several immature oogonia. Also present are ovoid structures, which may represent differently shaped oogonia, hyphal swellings, or zoosporangia. Small dome-shaped structures, probably zoospore cysts, are attached to numerous host cell walls. This discovery sheds new light on the morphology and biology of water molds in a Carboniferous ecosystem.     

Metabolic isotopomer labeling systems. Part II: structural flux identifiability analysis

Metabolic flux analysis using carbon labeling experiments (CLEs) is an important tool in metabolic engineering where the intracellular fluxes have to be computed from the measured extracellular fluxes and the partially measured distribution of 13C labeling within the intracellular metabolite pools. The relation between unknown fluxes and measurements is described by an isotopomer labeling system (ILS) (see Part I [Math. Biosci. 169 (2001) 173]). Part II deals with the structural flux identifiability of measured ILSs in the steady state. The central question is whether the measured data contains sufficient information to determine the unknown intracellular fluxes. This question has to be decided a priori, i.e. before the CLE is carried out. In structural identifiability analysis the measurements are assumed to be noise-free. A general theory of structural flux identifiability for measured ILSs is presented and several algorithms are developed to solve the identifiability problem. In the particular case of maximal measurement information, a symbolical algorithm is presented that decides the identifiability question by means of linear methods. Several upper bounds of the number of identifiable fluxes are derived, and the influence of the chosen inputs is evaluated. By introducing integer arithmetic this algorithm can even be applied to large networks. For the general case of arbitrary measurement information, identifiability is decided by a local criterion. A new algorithm based on integer arithmetic enables an a priori local identifiability analysis to be performed for networks of arbitrary size. All algorithms have been implemented and flux identifiability is investigated for the network of the central metabolic pathways of a microorganism. Moreover, several small examples are worked out to illustrate the influence of input metabolite labeling and the paradox of information loss due to network simplification.     

Ion homeostasis and apoptosis.

Alterations in the transmembrane gradients of several physiological ions may influence programmed cell death. In particular, recent data suggest that increases in intracellular calcium may either promote or inhibit apoptosis, depending on the level, timing and location, whereas loss of intracellular potassium promotes apoptosis.     

Congenital disorders of vitamin B12 transport and their contributions to concepts. II.

Congenital deficiencies of Transcobalamin II (TC II) and R binders of vitamin B12 (B12, cobalamin, Cbl) have been described in several families. The deficiency of TC II exists as at least three variants. The deficiency of TC II is expressed by a profound megaloblastic pancytopenia during the first few weeks of life, but the serum Cbl is normal. In contrast, the deficiency of R binder is asymptomatic, tissues are replete in Cbl, but the serum Cbl is low. All of the R binder in the several body sources is under the same genetic control. Studies of the congenital deficiency TC II suggest the following: (1) The function of TC II is the promotion of cell uptake of physiologic amounts of Cbl, which can also be accomplished by very large amounts of Cbl, and not in any intracellular process. (2) TC II is essential for the absorption, postabsorptive distribution, and recycling of TC II. (3) The metabolic consequences of TC II deficiency are expressed primarily in rapidly dividing cells probably because they are dependent upon the constant need for new Cbl.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an intracellular parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other filamentous brown algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Determination of intracellular osmotic volume and sodium concentration in dunaliella

A new method to measure intracellular volume in Dunaliella was developed, where lithium ions are used as monitors of the extracellular volume. Li⁺ is shown to be impenetrable to the intracellular volume, insignificantly absorbed to the algae, and is rapidly and evenly distributed within the extracellular volume. The method is suggested to be free of several limitations and consistent errors present in several previously employed techniques.

Using the new technique it is shown that both Dunaliella salina and Dunaliella bardawil adjust to a constant cellular volume when grown in a medium containing salt concentrations ranging from 0.5 molar to 4 molar NaCl. That volume is 90 femtoliter per cell for D. salina and 600 femtoliter per cell for D. bardawil. Nonosmotic volume accounts for about 10% of the total cell volume.

The intracellular sodium concentration, as determined with the new technique, was under all experimental conditions tested below 100 millimolar. This was true both for cells grown on 0.5 to 4 molar NaCl, and during the osmoregulatory process. It is thus concluded that intracellular NaCl is a minor contributor to the overall intracellular osmotic pressure in Dunaliella.

     

Induction of filopodia by direct local elevation of intracellular calcium ion concentration.

In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca2+]i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+]i increased from approximately 60 nM to approximately 1 microM within the illuminated zone, and then returned to resting level in approximately 10-15 s. New filopodia arose in this area within 1-5 min, and persisted for approximately 15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca2+]i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia.     

Recombinant viruses as tools to induce protective cellular immunity against infectious diseases.

Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms. Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen. However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes. In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools. Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes. This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development. Thus, this review will focus in particular on the development of new vaccination protocols against these diseases.     

Ionic, electrical and water balance in the animal cell. The system with active cation transport, Goldman's channels and the Na + K +2Cl-type symport]

The relations between intracellular potassium, sodium water content and resting potential on the one hand and the ion transport parameters and intracellular electrical charge on the other hand were computed for a model of animal cell with a several ion transporters and variable intracellular charge. The case of the balanced ion distribution is considered. The results are presented in a graphical form.     

Biological ion exchanger resins. VII. Counter-ion activity coefficients in solutions of biological polyelectrolytes.

The single ion activity coefficients of K+ and Cl- counter-ions were determined in concentrated polyelectrolyte solutions. The polyelectrolytes investigated included DNA and several proteins. Results indicate that ion gradients of up to 40:1 do not lower the counter-ion activity coefficient below 0.5. Thus, published values of the intracellular activity coefficient of K+ are not incompatible with cellular models utilizing cytoplasmic ion exchange.     

Electrophysiological recording from brain slices.

This article discusses several of the currently used methodologies for recording from brain slices. Aspects of slice preparation as well as appropriate uses for the various slice models (i.e., thin or thick slices) are considered. The merits of extracellular and intracellular electrophysiological recording and their uses are discussed. In addition, mechanisms of neuronal circuit activation and stimulation are presented.     

Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane

It is assumed that the survival factors Bcl-2 and Bcl-x(L) are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-x(L) is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-x(L) requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-x(L) specifically functions on the MOM.     

Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress.

Polyols, or polyhydroxy alcohols, are produced by many fungi. Saccharomyces cerevisiae produces large amounts of glycerol, and several fungi that cause serious human infections produce D-arabinitol and mannitol. Glycerol functions as an intracellular osmolyte in S. cerevisiae, but the functions of D-arabinitol and mannitol in pathogenic fungi are not yet known. To investigate the functions of mannitol, we constructed a new mannitol biosynthetic pathway in S. cerevisiae. S. cerevisiae transformed with multicopy plasmids encoding the mannitol-1-phosphate dehydrogenase of Escherichia coli produced mannitol, whereas S. cerevisiae transformed with control plasmids did not. Although mannitol production had no obvious phenotypic effects in wild-type S. cerevisiae, it restored the ability of a glycerol-defective, osmosensitive osg1-1 mutant to grow in the presence of high NaCl concentrations. Moreover, osg1-1 mutants producing mannitol were more resistant to killing by oxidants produced by a cell-free H2O2-FeSO4-NaI system than were controls. These results indicate that mannitol can (i) function as an intracellular osmolyte in S. cerevisiae, (ii) substitute for glycerol as the principal intracellular osmolyte in S. cerevisiae, and (iii) protect S. cerevisiae from oxidative damage by scavenging toxic oxygen intermediates.     

Spiral waves and intracellular calcium signalling.

Confocal imaging of intracellular Ca2+ brings a new level of resolution to the study of hormonal control of intracellular Ca2+ release. This approach has demonstrated the existence of pulsatile circular and spiral waves of Ca+ release induced by receptor activation. The data obtained by confocal imaging support a new framework for understanding intracellular Ca2+ signalling. The goal of this chapter is to review our data on the complexity of intracellular Ca2+ release in Xenopus oocytes, introduce the concept of Ca2+ excitability as a model for Ca2+ release and discuss the implications for encoding intracellular signal information.     

Tyrosine phosphorylation induced by C4 peptide constructs from HIV Gp120.

Treatment of HUT78 cells with CD4-binding peptide constructs derived from the C4 domain of HIV-1 gp120 results in autophosphorylation of a src-related kinase, p56lck. This leads to p56lck activation and the subsequent phosphorylation of tyrosine residues in several intracellular proteins. The phosphorylation is specific to the C4 peptides as no new phosphorylation occurs when the cells are treated with control peptides or polymers. The induction of tyrosine phosphorylation by the C4 peptide constructs depends on the capability of the peptide to assume a helical conformation because similar peptide constructs that were not able to form helices did not induce cellular tyrosine phosphorylation.