Neuronal calcium sparks and intracellular calcium “noise”

Intracellular calcium ions are involved in many forms of cellular function. To accommodate so many control functions, a complex spatiotemporal organization of calcium signaling has developed. In both excitable and nonexcitable cells, calcium signaling was found to fluctuate. Sudden localized increases in the intracellular calcium concentration—or calcium sparks—were found in heart, striated and smooth muscle, Xenopus Laevis oocytes, and HeLa and P12 cells. In the nervous system, intracellular calcium ions were found important in key processes such as transmitter release, repetitive firing, and gene expression. Hence, we examined whether calcium sparks also exist in neurons. Using confocal laser-scanning microscopy and fluorescent probes, we found that calcium sparks exist in two types of neuronal preparations: the presynaptic boutons of the lizard neuromuscular junction and rat hippocampal neurons in cell culture. Control experiments exclude the possibility that these calcium sparks originate from instrumental or biological artifacts. Calcium sparks seem to be just the tip of the iceberg of a more general phenomenon of intracellular calcium “noise.” We speculate that calcium sparks and calcium noise may be of key importance in calcium signaling in the nervous system.

Effective intercellular communication distances are determined by the relative time constants for cyto/chemokine secretion and diffusion

A cell’s ability to effectively communicate with a neighboring cell is essential for tissue function and ultimately for the organism to which it belongs. One important mode of intercellular communication is the release of soluble cyto- and chemokines. Once secreted, these signaling molecules diffuse through the surrounding medium and eventually bind to neighboring cell’s receptors whereby the signal is received. This mode of communication is governed both by physicochemical transport processes and cellular secretion rates, which in turn are determined by genetic and biochemical processes. The characteristics of transport processes have been known for some time, and information on the genetic and biochemical determinants of cellular function is rapidly growing. Simultaneous quantitative analysis of the two is required to systematically evaluate the nature and limitations of intercellular signaling. The present study uses a solitary cell model to estimate effective communication distances over which a single cell can meaningfully propagate a soluble signal. The analysis reveals that: (i) this process is governed by a single, key, dimensionless group that is a ratio of biological parameters and physicochemical determinants; (ii) this ratio has a maximal value; (iii) for realistic values of the parameters contained in this dimensionless group, it is estimated that the domain that a single cell can effectively communicate in is ≈250 μm in size; and (iv) the communication within this domain takes place in 10–30 minutes. These results have fundamental implications for interpretation of organ physiology and for engineering tissue function ex vivo.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix–loop–helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia.

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

Visualization of biochemical networks in living cells

Functional annotation of novel genes can be achieved by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. We report an experimental strategy that allows for detection of protein interactions and functional assays with a single reporter system. Interactions among biochemical network component proteins are detected and probed with stimulators and inhibitors of the network. In addition, the cellular location of the interacting proteins is determined. We used this strategy to map a signal transduction network that controls initiation of translation in eukaryotes. We analyzed 35 different pairs of full-length proteins and identified 14 interactions, of which five have not been observed previously, suggesting that the organization of the pathway is more ramified and integrated than previously shown. Our results demonstrate the feasibility of using this strategy in efforts of genomewide functional annotation.

Historical overview: Searching for replication help in all of the rec places

For several decades, research into the mechanisms of genetic recombination proceeded without a complete understanding of its cellular function or its place in DNA metabolism. Many lines of research recently have coalesced to reveal a thorough integration of most aspects of DNA metabolism, including recombination. In bacteria, the primary function of homologous genetic recombination is the repair of stalled or collapsed replication forks. Recombinational DNA repair of replication forks is a surprisingly common process, even under normal growth conditions. The new results feature multiple pathways for repair and the involvement of many enzymatic systems. The long-recognized integration of replication and recombination in the DNA metabolism of bacteriophage T4 has moved into the spotlight with its clear mechanistic precedents. In eukaryotes, a similar integration of replication and recombination is seen in meiotic recombination as well as in the repair of replication forks and double-strand breaks generated by environmental abuse. Basic mechanisms for replication fork repair can now inform continued research into other aspects of recombination. This overview attempts to trace the history of the search for recombination function in bacteria and their bacteriophages, as well as some of the parallel paths taken in eukaryotic recombination research.

Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus.

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.

Impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis mice.

Cystic fibrosis is a disease characterized by abnormalities in the epithelia of the lungs, intestine, salivary and sweat glands, liver, and reproductive systems, often as a result of inadequate hydration of their secretions. The primary defect in cystic fibrosis is the altered activity of a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) channel. However, it is not clear how a defect in the CFTR Cl- channel function leads to the observed pathological changes. Although much is known about the structural properties and regulation of the CFTR, little is known of its relationship to cellular functions other than the cAMP-dependent Cl- secretion. Here we report that cell volume regulation after hypotonic challenge is also defective in intestinal crypt epithelial cells isolated from CFTR -/- mutant mice. Moreover, the impairment of the regulatory volume decrease in CFTR -/- crypts appears to be related to the inability of a K+ conductance to provide a pathway for the exit of this cation during the volume adjustments. This provides evidence that the lack of CFTR protein may have additional consequences for the cellular function other than the abnormal cAMP-mediated Cl- secretion.

Pré-condicionamento isquêmico remoto em portadores de claudicação intermitente de membros inferiores

INTRODUÇÃO: Pré-condicionamento isquêmico remoto (PCIR) é o fenômeno pelo qual curtos períodos de isquemia sub-letal sobre um órgão ou tecido, intercalados com reperfusão do mesmo, conferem a outros órgãos ou tecidos distantes deste, um aumento na capacidade da resistir a episódios subsequentes de isquemia, a qual os mesmos possam ser expostos. Com base nesse fato, testamos a hipótese de que o pré-condicionamento isquêmico remoto em pacientes portadores de claudicação intermitente de membros inferiores poderia aumentar a capacidade de deambulação desses pacientes, extrapolando o conceito do PCIR de aumento da capacidade de preservação da integridade celular frente à isquemia, para a manutenção da função celular, tornando a célula mais apta ao trabalho em situações de privação de oxigênio, geradas pela restrição do fluxo sanguíneo, como ocorre nos pacientes com claudicação intermitente de membros inferiores, durante o exercício. OBJETIVOS: Avaliar se o PCIR aumenta a distância de início de claudicação e/ou a distância total de claudicação em pacientes com doença arterial obstrutiva periférica. MÉTODOS: Foram estudados 52 pacientes ambulatoriais que apresentavam queixa de claudicação intermitente dos membros inferiores, associada a um pulso arterial ausente ou reduzido no membro sintomático e/ou um índice tornozelo-braço <0,90. Estes pacientes foram randomizados em três grupos (A, B e C). Todos os pacientes foram submetidos a dois testes de caminhada em esteira de acordo com o protocolo de Gardner. O grupo A fez o primeiro teste de esteira sem o pré-condicionamento isquêmico remoto e, após 7 dias, foi submetido a um novo teste de esteira, agora precedido pelo pré-condicionamento isquêmico remoto. O grupo B foi submetido ao pré-condicionamento isquêmico remoto antes do primeiro teste de esteira e, após 7 dias, realizou novo teste de esteira, agora sem o pré-condicionamento isquêmico remoto. Já no Grupo C (grupo controle), ambos os testes de esteira foram realizados sem pré-condicionamento isquêmico remoto, também com 7 dias de intervalo. RESULTADOS: Os grupos A e C mostraram um aumento na distância de início de claudicação, no segundo teste, em comparação com o primeiro teste. O grupo A teve um aumento estatisticamente significativo, em relação ao grupo C (grupo controle). Com relação à distância total de claudicação, todos os grupos (A, B e C), mostraram um aumento estatisticamente significativo a favor do segundo teste, porém não foi observada diferença entre os grupos (A, B e C). CONCLUSÕES: O pré-condicionamento isquêmico remoto aumentou a distância inicial de claudicação em pacientes com claudicação intermitente, no entanto, ele não afetou a distância total de claudicação dos pacientes portadores de claudicação intermitente de membros inferiores

Menin associates with a trithorax family histone methyltransferase complex and with the Hoxc8 locus

The cellular function of the menin tumor suppressor protein, product of the MEN1 gene mutated in familial multiple endocrine neoplasia type 1, has not been defined. We now show that menin is associated with a histone methyltransferase complex containing two trithorax family proteins, MLL2 and Ash2L, and other homologs of the yeast Set1 assembly. This menin-associated complex methylates histone H3 on lysine 4. A subset of tumor-derived menin mutants lacks the associated histone methyltransferase activity. In addition, menin is associated with RNA polymerase II whose large subunit carboxyl-terminal domain is phosphorylated on Ser5. Men1 knockout embryos and cells show decreased expression of the homeobox genes Hoxc6 and Hoxc8. Chromatin immunoprecipitation experiments reveal that menin is bound to the Hoxc8 locus. These results suggest that menin activates the transcription of differentiation-regulating genes by covalent histone modification, and that this activity is related to tumor suppression by MEN1.

The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

Lipid rafts and plasma membrane microorganization: insights from Ras

The spatial organization of plasma membrane components in discrete microdomains is thought to be a key factor in the generation of distinct signal outputs. A detailed characterization of plasma membrane microdomains, including descriptions of their size, dynamics and abundance, has proved to be a taxing problem for cell biologists and biophysicists. The use of novel techniques is providing exciting new insights into the challenging problem of plasma membrane microstructure and has allowed the visualization of domains with the characteristics expected of lipid rafts - microdomains of the plasma membrane enriched in cholesterol and sphingolipids. This review focuses on some of these recent advances and uses Ras signaling as a paradigm for understanding inner plasma membrane organization and the role of lipid rafts in cellular function.

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.