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Molecular events associated with acquisition of heat tolerance by the yeast Saccharomyces cerevisiae.

The heat shock response is an inducible protective system of all living cells. It simultaneously induces both heat shock proteins and an increased capacity for the cell to withstand potentially lethal temperatures (an increased thermotolerance). This has lead to the suspicion that these two phenomena must be inexorably linked. However, analysis of heat shock protein function in Saccharomyces cerevisiae by molecular genetic techniques has revealed only a minority of the heat shock proteins of this organism having appreciable influences on thermotolerance. Instead, physiological perturbations and the accumulation of trehalose with heat stress may be more important in the development of thermotolerance during a preconditioning heat shock. Vegetative S. cerevisiae also acquires thermotolerance through osmotic dehydration, through treatment with certain chemical agents and when, due to nutrient limitation, it arrests growth in the G1 phase of the cell cycle. There is evidence for the activities of the cAMP-dependent protein kinase and plasma membrane ATPase being very important in thermotolerance determination. Also, intracellular water activity and trehalose probably exert a strong influence over thermotolerance through their effects on stabilisation of membranes and intracellular assemblies. Future investigations should address the unresolved issue of whether the different routes to thermotolerance induction cause a common change to the physical state of the intracellular environment, a change that may result in an increased stabilisation of cellular structures through more stable hydrogen bonding and hydrophobic interactions.

Adenosine Triphosphatases↗

The evolution of development in Streptomyces analysed by genome comparisons.

There is considerable information about the genetic control of the processes by which mycelial Streptomyces bacteria form spore-bearing aerial hyphae. The recent acquisition of genome sequences for 16 species of actinobacteria, including two streptomycetes, makes it possible to try to reconstruct the evolution of Streptomyces differentiation by a comparative genomic approach, and to place the results in the context of current views on the evolution of bacteria. Most of the developmental genes evaluated are found only in actinobacteria that form sporulating aerial hyphae, with several being peculiar to streptomycetes. Only four (whiA, whiB, whiD, crgA) are generally present in nondifferentiating actinobacteria, and only two (whiA, whiG) are found in other bacteria, where they are widespread. Thus, the evolution of Streptomyces development has probably involved the stepwise acquisition of laterally transferred DNA, each successive acquisition giving rise either to regulatory changes that affect the conditions under which development is initiated, or to changes in cellular structure or morphology.

Bacterial Proteins↗

Scanning electron microscope observations on collagen fibers in human dentin and pulp.

Human permanent teeth were examined in the scanning electron microscope after demineralization and exposure to preparative procedures based on hydrogen peroxide, trypsin, and EDTA. These substances removed the inorganic material, the cellular structures, the homogeneous connective tissue ground substance, and interfibrillar matrix. The remaining tissue components comprised a network of distinct collagen fibers whose organization was related to the type of tissue in which these were incorporated. A similar or identical method has not been developed or applied to teeth previously. Dentin and predentin comprised a compact mass of fibers which basically were parallel to the continuously growing interior surface of the predentin, or arranged at an acute angle to this plane. Collagen fibers in the pulp were numerous, but lacked any particular orientation in most areas. Interodontoblastic fibers crossed the odontoblastic zone at a right angle to the pulp chamber wall and mingled with collagen fibers in predentin. When previously published findings of ours are taken into account, it is possible to conclude that other factors than the organization of the collagen fibers are responsible for the stainability of these fibers in predentin and in interglobular dentin with silver methenamine, and that aldehyde groups on collagen fibers in predentin may be actively and directly involved in the mineralization of the dentin.

Collagen↗

Cytoplasmic organization of POXvirus DNA replication.

Poxviruses, a family of large DNA viruses, are unique among DNA viruses, because they carry out DNA replication in the cytoplasm rather than the nucleus. This process does not occur randomly, but instead, these viruses create cytoplasmic 'mini-nuclei', distinct sites that are surrounded by membranes derived from the rough endoplasmic reticulum (ER) that support viral replication. This review summarizes how distinct steps preceding cytoplasmic DNA replication, as well as replication itself, operate in the host cell. The collective data point to an important role for both the rough ER and the microtubules and indicate that these cellular structures help to co-ordinate the virus life cycle to ensure that individual steps occur at the right time and place. In a broader sense, they emphasize how viruses have evolved sophisticated ways to use host cells to optimize their life cycles to ensure efficient production of infectious progeny.

Cytoplasm↗

Regulation of cytoskeletal dynamics at the immune synapse: new stars join the actin troupe.

Reorganization of actin cytoskeletal dynamics plays a critical role in controlling T-lymphocyte activation and effector functions. Interaction of T-cell receptors (TCR) with appropriate major histocompatibility complex-peptide complexes on antigen-presenting cells results in the activation of signaling cascades, leading to the accumulation of F-actin at the cell-cell contact site. This event is required for the formation and stabilization of the immune synapse (IS), a cellular structure essential for the modulation of T-cell responses. Analysis of actin cytoskeletal dynamics following engagement of the TCR has largely focused on the Arp2/3 regulator, WASp, because of its early identification and its association with human disease. However, recent studies have shown equally important roles for several additional actin regulatory proteins. In this review, we turn the spotlight on the expanding cast of actin regulatory proteins, which co-ordinate actin dynamics at the IS.

Actins↗

Studies on the rat liver following iron overload.

Sprague-Dawley rats were given Jectofer injections to obtain iron overload in rat liver Kupffer and parenchymal cells. The release of iron was studied on the light and electron microscopical level by bleeding of the iron loaded animals and comparing the results with non bled controls. During the mobilization of iron. Kupffer cells showed a rapid and almost complete disappearance of electron dense iron containing particles (IP) while there was only limited decline of IPs in the parenchymal cells. Iron was demonstrated with X-ray microanalysis at the ultrastructural level, and presence of acid phosphatase was revealed using a histochemical method based on precipitation of lead phosphate at sites of enzyme activity in combination with X-ray microanalysis. It was demonstrated that all IP-containing cytoplasmic bodies also showed presence of acid phosphatase implying that all of them represented lysosomes. The mechanism of release of iron from Kupffer and parenchymal cells was discussed with special emphasis on the role of the lysosomes and the relationship between their contents and other cellular structures.

Acid Phosphatase↗

New surgical approach for root coverage of localized gingival recession with acellular dermal matrix: a 12-month comparative clinical study.

BACKGROUND: Acellular dermal matrix graft (ADMG) has been used as an advantageous substitute for autogenous subepithelial connective tissue graft (SCTG). However, the surgical techniques used were primarily developed for the SCTG, and they may not be adequate for ADMG since it has a different healing process than SCTG owing to its different vascular and cellular structures. This study compared the 1-year clinical outcome of a new surgical approach with the outcome of a conventional procedure for the treatment of localized gingival recessions, both performed using the ADMG. MATERIALS AND METHODS: The clinical parameters-probing depth, relative clinical attachment level, gingival recession (GR), and width of keratinized tissue-of 32 bilateral Miller Class I or II gingival recessions were assessed at baseline and 12 months postoperatively. RESULTS: Significant clinical changes for both surgical techniques were achieved after this period, including GR reduction from 3.4 mm presurgery to 1.2 mm at 1 year for the conventional technique and from 3.9 mm presurgery to 0.7 mm at 1 year for the new technique. The percentage of root coverage was 62.3% and 82.5% for the conventional and new techniques, respectively. Comparisons between the groups after this period by Mann-Whitney rank sum test revealed statistically significant greater reduction of GR favoring the new procedure (p = .000). CLINICAL SIGNIFICANCE: Based on the results of this study, it can be concluded that a new surgical technique using an ADMG is more suitable for root coverage when compared with the conventional technique. The results revealed a statistically significant improvement in clinical performance with the ADMG approach.

Adult↗

Response of cytoskeleton of murine osteoblast cultures to two-step freezing.

Understanding the ultrastructural response of cells to the freezing process is important for designing cryopreservation strategies for cells and tissues. The cellular structures of attached cells are targets of cryopreservation-induced damage. Specific fluorescence staining was used to assess the status of the actin filaments (F-actin) of murine osteoblasts attached to hydroxyapatite discs and plastic coverslips for a two-step freezing process. The F-actin of dead cells was depolymerized and distorted in the freezing process, whereas that of live cells had little change. The results suggest that the cytoskeleton may support the robustness of cells during cryopreservation. The present study helps to investigate the damage mechanism of attached cells during the freezing process.

Actins↗

Basic aspects of calcium chemistry and membrane interaction: on the messenger role of calcium.

To summarize the perspective developed in this lecture we begin by considering it as axiomatic: (1) that aqueous domains delimited by lipid membranes typify cellular structure; (2) that different compositions of extracellular and intracellular aqueous domains and differences among intracellular aqueous domains require selective permeation of lipid membranes; and (3) that inorganic ion movements across lipid membranes are a common denominator in permeation. This set of axioms lead to the following set of postulates: (1) Evolutionary solutions to the problematic apposition of ions and lipid barriers are fundamental aspects of cell function. (2) An effective messenger role for communication between aqueous domains requires (a) meticulous modulation of movement across lipid membranes and (b) selective interactions within aqueous domains. (3) An ion would be an effective messenger. In a search for an appropriate ionic messenger it is noted that inorganic cations have a wider range of interactions with biomolecules than anions, that the prevalent monovalent cations have too high a flux across lipid membranes and too weak an interaction with molecules in aqueous domains, that trivalent cations cannot as effectively be transported across lipid membranes, that divalent cation movement across lipid membranes can be well modulated and their divalent charge allows for a wide range of binding constants with biological molecules, and that for reasons of radius-compatibility with polypeptide chelation and due to the lack of stringent crystal field requirements, Ca2+ is a most suitable divalent cation for a messenger role.

Anti-Bacterial Agents↗

Genomes and form. The case for teleomorphic recursivity.

The genotype-phenotype (genome-form) distinction is considered by many to be fundamental to modern evolutionary thinking. Indeed, the premises that: DNA solely constitutes the genotype; that the phenotype is a transient product of the genotype, with the latter not only describing, but also implementing the construction of the former; and that the constructed materials and systems of the cell have no impact on the genotype, have become dogmas. Yet a vast body of data from molecular genetics reveals that cellular systems, directly and indirectly, alter the genome. Some of these data are reviewed. Proteins can influence mutations along the chromosomes, heritably modify the information content of DNA sequences, and, in some instances, reorganize the germline or somatic genome via DNA engineering pathways. These data suggest that the constructed (proteins, chromatin arrays, and metabolic pathways) has an important role in shaping the descriptor. Insofar as it is biochemically possible for states adopted by cellular structures to be stabilized and eventually memorized by engineering chromosomes, semantic closure can be transcended--meaning can be transferred from the domain of form to the genome, and this presumably ongoing process is termed teleomorphic recursivity. Throughout the paper, I implicitly argue that the genome-form partition is strictly a formal one, with no deeply material basis.

Biological Evolution↗

Rickettsial actin-based motility: behavior and involvement of cytoskeletal regulators.

Actin-based motility (ABM) is employed by spotted fever group (SFG) rickettsiae, such as Rickettsia rickettsii, to promote cell-to-cell spread. Time-lapse video microscopy revealed that ABM is not strictly confined to SFG rickettsiae as typhus group R. typhi moved at approximately the same rate as R. rickettsii (approximately 4 micro m/min), but in a highly erratic fashion. A number of common behaviors were observed between ABM of R. typhi and R. rickettsii, such as entrance into plasma membrane protrusions, formation of new actin tails only on the old surface of newly formed daughter cells, and quick (within 15 sec) reassembly of the actin tail to the opposite pole upon contact with cellular structures that impede forward movement. This last behavior suggests that the rickettsial protein(s) required for ABM is uniformly localized to both poles of the bacterium and possibly throughout the rickettsial surface. Functional roles in rickettsial ABM for neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex, critical regulators of ABM of other pathogens, have not been established. Domains of N-WASP that have characterized inhibitory effects on N-WASP or Arp2/3 complex function were expressed in HeLa cells infected with R. rickettsii. Shigella flexneri-infected cells were used as a control. When ectopically expressed, the VCA domain of N-WASP (VCA) acts as a dominant/negative with respect to Arp2/3 complex function and N-WASP missing VCA (DeltaVCA) acts as a dominant/negative form of N-WASP. Expression of VCA or DeltaVCA severely inhibited S. flexneri ABM (no Shigella motility observed in the majority of expressing cells) while only moderately inhibiting ABM of R. rickettsii (approximately 35% decrease in the rate of ABM). In addition, ectopically expressed full-length GFP-N-WASP was recruited by S. flexneri but not R. rickettsii, and Arp3 was detected by indirect immunofluorescence in S. flexneri actin tails but not within R. rickettsii actin tails. Collectively, these data suggest that rickettsial ABM is independent of N-WASP and Arp2/3 complex function.

Actins↗

A study of virus-like particles present in bovine nerve sheath tumours.

The morphology of virus-like particles present in bovine nerve sheath tumours is described, and their origin and significance discussed. Virus-like particles were investigated using transmission electron microscopy and tissue culture. The particles were found in a number of tumours and were present either as aggregates in pseudonuclear inclusions or as budding forms associated with the plasma membrane. The latter were found only in cultured tissue. Although other cellular structures were considered it was thought that the particles represented virus, with those in pseudonuclear inclusions being an intermediate form. Both bovine syncytial virus and oncovirinae were considered as possibilities in the aetiology of the condition.

Animals↗

Computerized morphometry of mean nuclear diameter and nuclear roundness in canine mammary gland tumors on cytologic smears.

BACKGROUND: The use of computer-based image analysis systems in veterinary oncology has increased. Computerized morphometry is a part of image analysis that describes geometric figures of cellular structures in any dimension. Most investigators have performed morphometric analysis on histologic specimens. Computer-assisted nuclear cytomorphometry can provide important preoperative information on neoplastic lesions in animals. OBJECTIVES: The aim of this study was to define whether the morphometric parameters of mean nuclear diameter and nuclear roundness could be used to differentiate benign from malignant canine mammary gland tumors on cytologic specimens. METHODS: Mean nuclear diameter and nuclear roundness were determined by computer-assisted morphometry of epithelial cells in Hemacolor-stained cytologic smears from normal canine mammary gland (n = 7) and from canine mammary adenomas (n = 8), tubulopapillary carcinomas (n = 9), and solid carcinomas (n = 6). Data were analyzed by the Mann-Whitney U test. RESULTS: Significant differences (P <.001) were found in mean nuclear diameter and nuclear roundness among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for nuclear roundness between tubulopapillary carcinomas and solid carcinomas). CONCLUSIONS: The morphometric parameters of mean nuclear diameter and nuclear roundness can be used in the preoperative differentiation of benign from malignant canine mammary gland tumors.

Animals↗

Paracetamol confers resistance to ethanol-induced gastric mucosal damage in rats.

Paracetamol given orally or subcutaneously did not produce any observable gastric mucosal damage, nor did it change the acidity of the residual secretion in rat stomachs. However, it delayed the gastric emptying rate and increased the residual volume of gastric secretion. Pretreatment with paracetamol 250 mg kg-1 significantly prevented ethanol-induced gastric ulceration. Although it did not influence ethanol-stimulated acid secretion, it increased the mucosal mucus content in the ethanol-treated animals. The findings suggest that the protective mechanism of paracetamol against ethanol-induced damage is likely to be due to improved gastric mucosal integrity, through an increase in the adherent mucosal mucus which protects the underlying delicate cellular structures.

Acetaminophen↗

The G. L. Brown lecture. Adventures in anaesthesia.

The results presented in this lecture show that anaesthetic agents impede the transfer of information from the periphery to the cerebral cortex. This is shown both as a reduction in the amplitudes of the initial positive and negative waves of the cerebral cortical response evoked by simulation of the periphery and as an increase in the latency of this response. This effect is most probably a prime effect of anaesthesia since (a) it is common to all the anaesthetics used, (b) the potency of the anaesthetics is directly proportional to their lipid solubility, and (c) the effect is reversed by high ambient pressures. The major site at which information transfer is most susceptible to the action of anaesthetics is at the level of the ventrobasal thalamus, although the cells in cortical layer V also appear to have an enhanced susceptibility to anaesthetic action. This latter observation is seen both in whole animal and cortical slice preparations. None the less, the first site of synaptic transfer at which anaesthetics exert a profound effect is upon the monosynaptically generated responses of ventrobasal thalamic neurones to cuneothalamic input. A possible mechanism of action for anaesthetic agents acting at this site would be upon a hypothetical cortico-thalamic-reticular-thalamic loop with the theoretical ability to control the responsiveness of the ventrobasal thalamic cells. This action was proposed both from the activity of neurones in response to anaesthetic agents and the anatomical arrangement seen in the thalamus. The thalamic reticular nucleus is a curved sheath of cells situated between the internal capsule and the external medullary lamina, capping and bounding laterally the specific nuclei of the dorsal thalamus. There is both anatomical and physiological evidence that the thalamic reticular nucleus comprises part of the thalamic reticular formation: its cellular structure also resembles that of the brain stem regions of the reticular formation (Ramon-Moliner, 1975). Early degeneration and Golgi studies showed that ascending fibres from the medial parts of the pontine and mesencephalic components of the brain stem reticular formation innervated the thalamic reticular nucleus ventrally, by penetrating the zona incerta, and dorsally, via the intralaminar and dorsal thalamic nuclei (Scheibel & Scheibel, 1958). These observations have been confirmed and extended more recently and it appears that the major innervation of the thalamic reticular nucleus occurs via the ventral route which follows the entire course of the reticular nucleus. No fibres ascending from the dorsal column lemniscal system, the spino-cervico-lemniscal system or the spinothalamic tract have been observed to terminate within the thalamic reticular nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Anesthesia↗

Voltage-dependent activation of potassium current in Helix neurones by endogenous cellular calcium.

1. The effect of endogenous Ca on potential-dependent K current IKD, was examined in identifiable neurones of Helix aspersa. The suction pipette method of internal perfusion was used along with a combined voltage-clamp method in which the membrane potential was measured by a separate glass micro-electrode and the current was passed by the suction pipette. Activation of the potential-dependent A current, IA, was prevented by using holding potentials of -40 mV where IA is inactivated and by the addition of the A-current blocker 4-aminopyridine. Activation of K currents by transmembrane Ca current, IKCa, was suppressed by Co substitution for Ca ion extracellularly. 2. Under these conditions, IKD rose to a peak value and then subsided to a steady level. The current-voltage (I-V) relationship for peak IKD had an upward bump at about +50 mV that gave it an S-shape. The I-V curve for steady IKD rose continuously. Peak and steady IKD were reduced by perfusing with EGTA or F ions intracellularly. The EGTA effect occurred at intracellular Ca activity levels below 10(-7) M. Increases in the concentration of EGTAi at constant Cai had no additional effect; however, recovery experiments do not allow us to rule out some direct action of EGTA on IKD. 3. Prolonged extracellular perfusion with Co-substituted solutions also reduced IKD and the effects occurred more quickly when the solutions were made hypertonic or caffeine was added to them. The peak transient was abolished, and the small remaining steady IKD (about 5-10% of normal peak IKD) was blocked by tetraethylammonium. IKD could be restored by the temporary reintroduction of Ca in the extracellular solution. 4. The S-shape of the peak I-V relationship for IKD may be due to Ca released from an endogenous site by membrane depolarization. The reduction of steady and peak IKD to very low values by Ca chelators or prolonged perfusion with Ca-free solutions indicates that Cai is important for activation of these K channels. 5. Three cellular structures were identified in electron micrographs of freeze-fractured neurones that could be involved in potential-dependent endogenous Ca release. These were a restricted extracellularly space, an intracellular membrane system of endoplasmic reticulum that may be fused to the internal face of the plasma membrane (the subsurface cisterns of Henkart & Nelson, 1979), and intracellular vesicles that also may be fused to the plasma membrane.

Animals↗

A cellular model of lung elasticity.

The mechanics of the lung parenchyma is studied using models comprised of line members interconnected to form 3-D cellular structures. The mechanical properties are represented as elastic constants of a continuum. These are determined by perturbing each individual cell from a reference state by an increment in stress which is superimposed upon the uniform stretching forces initially present in the members due to the transpulmonary pressure. A force balance on the distorted structure, together with a force-deformation law for the members, leads to a calculation of the strain increments of the members. Predictions based on the analysis of the 3-D isotropic dodecahedron are in good agreement with experimental values for the Young's, shear, and bulk moduli reported in the literature. The model provides an explanation for the dependence of the elastic moduli on transpulmonary pressure, the geometrical details of the structure, and the stress-strain law of the tissue.

Elasticity↗

Mechanism of photodynamic activity of pheophorbides.

Plasmid DNA is efficiently photocleaved by sodium pheophorbides (Na-Phdes) a and b in the absence of oxygen as well as in the presence of oxygen. Fluorescence microscopic observation shows a rapid incorporation of Na-Phde a into nuclei, mitochondria, and lysosome of human oral mucosa cells. In contrast Na-Phde b is incorporated only into the plasma membrane. The photodynamic activity of these pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures and involves two types of reactions: (i) direct electron transfer between DNA bases (especially guanine) and pheophorbide singlet excited state, and (ii) indirect reactions mediated by reactive oxygen species, including singlet oxygen whose production from molecular oxygen is sensitized by the Na-Phdes triplet state. A preliminary report has appeared in "Photodynamic Therapy of Cancer II," Proc. SPIE 2325, 416-424 (1994).

Chlorophyll↗