Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles.

Varicosities are ubiquitous neuronal structures that appear as local swellings along neurites of invertebrate and vertebrate neurons. Surprisingly little is known about their cell biology. We use here cultured Aplysia neurons and demonstrate that varicosities are motile compartments that contain large clusters of organelles. The content of varicosities propagate along neurites within the plasma membrane "sleeve", split and merge, or wobble in place. Confocal imaging, retrospective immunolabeling, electron microscopy and pharmacological perturbations reveal that the motility of the varicosities' organelle content occurs in concert with an actin scaffold and is generated by actomyosin motors. Despite the motility of these organelle clusters within the cytoplasm along the neurites, elevation of the free intracellular calcium concentration within varicosities by trains of action potentials induces exocytosis followed by membrane retrieval. Our observations demonstrate that varicosities formed in the absence of postsynaptic cells behave as "ready to go" prefabricated presynaptic terminals. We suggest that the varicosities' motility serves to increase the probability of encountering a postsynaptic cell and to rapidly form a functional synapse.

Analysis of genetic control elements in eukaryotes: transcriptional activity or nuclear hitchhiking?

A common way to analyse basal and stimulated activity of eukaryotic genetic control elements, such as promoters and enhancers, is to introduce them into cells via DNA vectors containing an easily assayable reporter gene. Activity is then studied by measurement of transiently produced mRNA or reporter protein. In such assays, it is assumed that the variable measured is proportional to the transcriptional activity of the control element under investigation. Here we question the validity of this generally accepted assumption. Specifically, recent observations indicate that control elements, in addition to modulating transgene transcription, can facilitate the nuclear uptake of their carrier plasmids. This process is mediated by transcription factors or other nuclear proteins harbouring nuclear localisation signals, which bind to the control elements in the cytoplasm and transport the DNA into the nucleus through the protein nuclear import machinery. As the number of mRNA transcripts produced for an epi-chromosomally expressed transgene is directly related to its copy number inside the nucleus, such transport activity may lead to substantial overestimation of the transcriptional potency of the control element(s) studied.

A regulated, NFkappaB-assisted import of plasmid DNA into mammalian cell nuclei.

The success of synthetic DNA delivery systems in human gene therapy will be enhanced by increasing transfection efficiencies and by providing tighter control over targeting of the DNA into the nucleus. Here, we used DNA vectors that contain repetitive binding sites for the inducible transcription factor NFkappaB, which is transported into the nucleus by the nuclear import machinery. Nuclear entry of the modified vectors was augmented 12-fold and was associated with corresponding increase in gene expression. Depending on their position, the binding sites could also function as transcriptional enhancers, increasing gene expression levels up to an additional 19-fold. Notably, nuclear targeting of the DNA and transgene transcription could both be regulated by exogenous stimulators which modulate the intracellular distribution of NFkappaB. The approach provides a framework for the controlled targeting of constitutive or transcriptionally regulated synthetic vectors into mammalian cell nuclei.

The Kaposi's sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1alpha.

The elucidation of the molecular mechanisms governing the transition from a nonangiogenic to an angiogenic phenotype is central for understanding and controlling malignancies. Viral oncogenes represent powerful tools for disclosing transforming mechanisms, and they may also afford the possibility of investigating the relationship between transforming pathways and angiogenesis. In this regard, we have recently observed that a constitutively active G protein-coupled receptor (GPCR) encoded by the Kaposi's sarcoma-associated herpes virus (KSHV)/human herpes virus 8 is oncogenic and stimulates angiogenesis by increasing the secretion of vascular endothelial growth factor (VEGF), which is a key angiogenic stimulator and a critical mitogen for the development of Kaposi's sarcoma. Here we show that the KSHV GPCR enhances the expression of VEGF by stimulating the activity of the transcription factor hypoxia-inducible factor (HIF)-1alpha, which activates transcription from a hypoxia response element within the 5'-flanking region of the VEGF promoter. Stimulation of HIF-1alpha by the KSHV GPCR involves the phosphorylation of its regulatory/inhibitory domain by the p38 and mitogen-activated protein kinase (MAPK) signaling pathways, thereby enhancing its transcriptional activity. Moreover, specific inhibitors of the p38 (SKF86002) and MAPK (PD98059) pathways are able to inhibit the activation of the transactivating activity of HIF-1alpha induced by the KSHV GPCR, as well as the VEGF expression and secretion in cells overexpressing this receptor. These findings suggest that the KSHV GPCR oncogene subverts convergent physiological pathways leading to angiogenesis and provide the first insight into a mechanism whereby growth factors and oncogenes acting upstream from MAPK, as well as inflammatory cytokines and cellular stresses that activate p38, can interact with the hypoxia-dependent machinery of angiogenesis. These results may also help to identify novel targets for the development of antiangiogenic therapies aimed at the treatment of Kaposi's sarcoma and other neoplastic diseases.

Thrombotic dysfibrinogenemia. Fibrinogen "Caracas V" relation between very tight fibrin network and defective clot degradability.

Fibrinogen Caracas V is a thrombotic dysfibrinogenemia with an Aalpha 532 Ser-->Cys mutation characterized by a tight fibrin network formed of thin fibers responsible for a less porous clot than a normal one. In the present work, fibrinogen Caracas V is further characterized in order to understand the relationship between the structural defect and thrombophilia. This thrombotic disorder has been attributed to a tight fibrin network responsible for a decreased permeation of flow through the clot, leading to defective thrombus lysis due to a diminished availability of fibrinolytic enzymes to the inner fibrin surface. Correction of clot structure anomaly, by addition of dextran 40 to fibrinogen before clotting, induces an improvement in fibrin degradation that was attributed to an increase in porosity. The pulmonary embolism observed in this family has been related to an hyper rigidity of the clot, an anomaly that is also corrected by dextran. Furthermore, this abnormal fibrinogen binds more albumin than does normal fibrinogen, a phenomenon attributed to the mutation of serine in Aalpha-532 by cysteine. Therefore, this fibrinogen shows a striking similarity to the fibrinogen Dusart, allowing us to confirm that the alphaC-terminal part of fibrinogen plays an important role in fibrin structure, and to conclude that the anomaly of fibrin network observed in fibrinogen Caracas V is responsible for a deficient thrombus lysis.

Tensin can induce JNK and p38 activation.

Cells organize diverse types of specialized adhesion sites upon attachment to extracellular matrix (ECM) components. One of the physiological roles of such cell-ECM interactions is to initiate and regulate adhesion-mediated signal transduction responses. The association of cells with fibronectin fibrils has been shown to regulate the JNK and p38 signaling pathways. We tested whether tensin, a cytoskeletal component localized to both focal contacts and fibronectin-associated fibrillar adhesions, can induce these signaling pathways. We found that tensin overexpression resulted in activation of both the c-Jun amino-terminal kinase (JNK) and p38 pathways. Tensin-mediated JNK activation was independent of the activities of the small GTP binding proteins Rac and Cdc42, but did depend on SEK, a kinase involved in the JNK pathway. We suggest that tensin may directly activate the JNK and p38 pathways, acting downstream or independent of the activities of the small GTP binding proteins Rac and Cdc42.

Dynamic beat-to-beat QT-RR relationship during physiotherapy effort in elderly patients without primary heart disease.

The ECGs from 18 patients hospitalized in a rehabilitation setting, following surgery for hip fracture, were examined to characterize the dynamic behavior of uncorrected QT interval in relation to changing RR interval during physiotherapy effort. ECG waveforms were analyzed to extract beat-to-beat QT and RR intervals using a computerized ECG Analyzer (CEA-1100). The method of defining the QT and RR intervals is based on performing multiple cross-correlations that enable rejection of artifacts from the analysis. The relationship between the RR and QT intervals was found using the following general formula QTi = cRRi-1b. Linear regression was performed on the logarithms of QT and RR measurements obtained to estimate the constant (a = log c) and the slope (b) values, reflecting the dynamic change of QT during physiotherapy effort. Having these two values, the dynamic QT extrapolated to a heart period of 1 second (QTcd) was calculated. The results were compared to the conventional corrected static QT according to the Bazzet formula (QTcs). The mean values of constants (a = log c) and slopes (b) over all patients were found to be 1.61 +/- 0.23 and 0.33 +/- 0.08, respectively, giving a QT (ms) heart-period (ms) dynamic relation of QTi = 41 x RR(i-1)0.33. The correlation between the dynamic QT and the static QT intervals was not significant. The mean values of the QTcd and QTcs intervals were significantly different (392 +/- 25 ms vs 434 +/- 28 ms; P < 0.0001). This dynamic measurement method of QT intervals may provide additional information on normal and abnormal cardiac repolarization in health and disease, helping in the diagnosis of cardiac disorders and arrhythmia risk.

A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho.

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho.

Symptomatic hypocalcemia precipitated by small-volume blood transfusion.

We present 2 cases in which the transfusion of small volumes of packed RBC was sufficient to precipitate symptomatic hypocalcemia. Subsequent inquiry revealed that both of the patients had preexisting, untreated, and asymptomatic hypocalcemia, 1 following partial thyroidectomy many years earlier and the other with documented hypocalcemia but without a definitive diagnosis.

Effector domain mutants of Rho dissociate cytoskeletal changes from nuclear signaling and cellular transformation.

The small GTP-binding Rho proteins control a variety of biological activities, including organization of the actin cytoskeleton, regulation of gene expression and cellular transformation. In contrast, Ras proteins do not induce actin stress fibers, but potently transform cells which exhibit a morphology clearly distinct from that caused by activated forms of Rho. To investigate whether nuclear signaling and oncogenic potential of Rho are a consequence of its profound effect on cytoskeletal organization, we replaced each amino acid in the Rho effector loop with those of Ras, or replaced conserved residues with others known to result in differential signaling capability when introduced into Ras and Rac1. These Rho mutants did not gain the ability to induce the MAPK, JNK or p38 pathways but, surprisingly, all Rho effector loop mutants still continued to induce actin stress fiber formation. However, three of these Rho mutants, with substitutions of leucine-39, glutamic acid-39, or cysteine-42, lost the ability to stimulate gene transcription via the serum response factor (SRF) and failed to induce neoplastic transformation. Thus, these results indicate that cytoskeletal changes are not sufficient to induce the transformed phenotype, and that Rho-effector molecules regulating the actin cytostructure are distinct from those signaling to the nucleus and subverting normal growth control.

Police attitudes toward mental illness and psychiatric patients in Israel.

The attitude of the public toward mental illness and toward psychiatric patients raises a serious and sensitive issue that indirectly affects the development of community mental health services. Most citizens feel that there is an association between mental illness and dangerous or violent behavior. Studies undertaken among police personnel in the 1970s demonstrated that their attitudes were similar to those of the general public in Israel. The objective of the present study was to assess the attitudes of police officers toward mental illness and psychiatric patients by means of a self-report questionnaire. Ninety-three policemen from five police stations within the Y. Abarbanel Mental Health Center catchment area participated in the study. All were young males (average age 32.1 years) and 75 percent had a high school education or higher. More than half (54.5%) had personally known a psychiatric patient in the past, and 20.4 percent of the police personnel graded mental illness as the severest form of disease in medicine. A minority (14.3%) of policemen agreed with the statement: "A psychiatric hospital should be fenced and manned by guards." One-third did not know whether psychiatric patients are dangerous. We conclude that training of police officers is called for to effect changes in their misconceptions about psychiatric patients. Psychoeducation may lead to improved handling by the police of incidents involving the mentally ill.

Forensic psychiatric service in Israel: ten years experience in an in-patient hospital unit.

In 1991 legislators revised the Mental Health Act in Israel, placing responsibility for forensic psychiatric evaluations with the district psychiatrist. The aim of the present paper is to describe the changing patterns of the forensic service in Israel's largest psychiatric hospital in the light of changing legislation. In the last 15 years a psychiatric forensic team provided evaluations both in an ambulatory clinic and a special in-patient ward. All medical records of subjects referred for forensic evaluations between 1982-1992 were re-examined. Demographic, forensic and psychiatric data were recorded. During this period the service experience a 15% increase in the number of evaluations. The team tended to prefer ambulatory evaluations. During the latter part of the period studied more referrals were with a previous criminal record and less with a history of mental disorders. An increase in drug and sex crimes was noted with a corresponding decrease in property and financial offences.

cDNA cloning and sequence analysis of the bovine adrenocorticotropic hormone (ACTH) receptor.

We isolated five independent cDNAs of nearly 3000 bp for the bovine ACTH receptor by screening adrenal cortex cDNA libraries with a PCR cloned cDNA fragment. The deduced receptor sequence includes 297 residues (M(r) = 33,258) with 81% identity with the human ACTH receptor, and shows seven hydrophobic transmembrane domains. The calculated M(r) of the receptor is smaller than the 40-45 kDa observed in crosslinking studies with labeled ACTH. Since the bovine and human receptors have two glycosylation motifs in the N-terminus, the difference may result from glycosylation of the receptor. Analysis of the sequences of both bovine and human receptors revealed a single protein kinase. A phosphorylation motif located in the third intracellular loop (Ser-209) juxtaposed to a protein kinase C phosphorylation motif (Thr-204). Thus, the involvement of protein kinase A and C pathways in ACTH action may be mediated in part by phosphorylation of the ACTH receptor at these motifs. The 3'-untranslated region of the bovine cDNA is > 2000 bp and includes two inverse repeats giving an extensive and strong secondary structure to the ACTH receptor RNA.

Signaling mechanisms controlled by melanocortins in melanoma, lacrimal, and brain astroglial cells.

Melanocortins appear to be involved as regulators in an ever growing number of physiological processes in cells and tissues of diverse functions. While such trends are apparent also in the case of other peptide hormones, it appears that melanocortin receptors can be regarded as unique among G-protein-linked receptors due to their special need for extracellular Ca2+ which may relate to some, yet undetermined selectivity of their actions. The physiological role that Ca2+ may be playing and the diverse signaling mechanisms regulated, as well as the nature of the cell-specific responses elicited in melanocortin-sensitive cells/tissues, have yet to be elucidated. Likewise, it will be of interest to establish the relationship of melanocortins to processes like growth and differentiation of cells, as well as to higher, more complex processes such as those regulated in the CNS.

Mechanism of action of melanocortin peptides. Possible role in astrocyte regulation.

Melanocortin peptides exert pleiotropic effect in numerous cell types, controlling processes ranging from adrenal steroidogenesis and melanocyte pigmentation to lacrimation and nerve regeneration. The binding of melanocortins to specific cell surface receptors initiates cellular responses via GTP binding proteins (G-proteins). The affinity of these peptides to the receptor is modulated by extracellular Ca2+ ions, a property unique to melanocortin receptors. In astrocyte cultures derived from the rat brain, melanocortin stimulation elevates cAMP levels that appear to induce morphological changes. However, a transient proliferative response to melanocortins in these cells appears to be cAMP independent. The presence of melanocortin receptors in brain tissue and their unique Ca2+ dependence are discussed in relation to their putative role as regulators of astrocytes.

Melanocortins stimulate proliferation and induce morphological changes in cultured rat astrocytes by distinct transducing mechanisms.

Melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and several peptides derived from pro-opiomelanocortin, are present in the dorsolateral hypothalamus and arcuate nucleus of several vertebrate species. These peptides affect central nervous system (CNS) functions including behavior, memory, and foetal brain development. In this study we investigated the effects of ACTH1-24, ACTH1-17, ACTH4-10, alpha-MSH, beta-MSH, and a potent analog (Nle4,D-Phe7)-alpha-MSH (melanocortins) on immunocytochemically defined astroglial cells prepared from primary cultures of 1-2-day-old rat brains. A cyclic adenosine 3',5'-monophosphate (cAMP) response to the melanocortins was only detected in astrocytes and not in other cell types in the culture. The extent of the cAMP response was greatest on day 21, the latest time tested. On the other hand, (methyl3H)-thymidine incorporation in astrocytes was significantly stimulated (1.5-2-fold) by melanocortins only in 7 and not in 14 and 21 day cultures. This mitogenic activity of melanocortins was not mimicked by other agents such as forskolin or isoproterenol which efficiently stimulate cAMP production in astrocytes. ACTH1-17 as a melanocortin representative induced significant morphological changes in 7 and 14 day cultures which included rounding of the cell body and process extension. This response, however, resembled that induced by forskolin and hence appears to be cAMP mediated. These findings suggest that astrocytes in the CNS may serve as a target for melanocortins. These peptides appear to affect differentiation and proliferation of these cells during certain developmental periods. While the morphological effects of melanocortins seem to be cAMP mediated, induction of proliferation of the astrocytes by melanocortins appears to involve an alternative signal transduction pathway.

Cortisol, ACTH, and beta-endorphin after dexamethasone administration in Parkinson's dementia.

The dexamethasone suppression test (DST) has been suggested as an effective tool for differentiating between depression and dementia. After administering 1 mg dexamethasone, we measured cortisol, ACTH, and beta-endorphin levels in 32 nondepressed patients with idiopathic Parkinson's disease (PD) (14 also with dementia) and 20 healthy, age-matched controls. Four of the 20 controls, 9 of the 18 with PD alone, and 8 of the 14 with PD and dementia were dexamethasone nonsuppressors (cortisol value greater than or equal to 5 micrograms/100 ml). PD patients without dementia (nonsuppressors) showed higher basal plasma values of cortisol (22.06 +/- 5.30 micrograms/100 ml) compared with the suppressors (13.38 +/- 3.30 micrograms/100 ml). Plasma ACTH and beta-endorphin responded in a coupled way to dexamethasone challenge. Higher basal levels of both peptides were found among PD patients (demented and nondemented), nonresponders to DST. Thus, the DST does not appear to be effective in differentiating between depression and dementia in PD. In addition, PD nonsuppressors showed higher basal values of plasma ACTH, beta-endorphin, and cortisol (similar to patients with major depression). This suggests that although the depression is clinically undetectable, both disorders may share some pathophysiological features at the hypothalamic hypophyseal adrenal level.

Lethal graft-versus-host reaction against non-H-2 antigens. I. Prevention of GVH-associated immunodeficiency by preimmunizing the donor against host-specific non-H-2 antigens.

Injection of B10.D2 cells into irradiated H-2d compatible (DBA/2xB10.D2)F1 recipients provokes a lethal GVH that can be abrogated by donor preimmunization against host-specific DBA/2 non-H-2 antigens. To study the possible relationship between the observed protection and restoration of immune responsiveness, we compared spleen cellularity, selected T and B cell functions, and NK activity in GVH and protected mice during the 1st month after grafting. Normal and isografted mice served as controls. GVH was found to be characterized by an early stimulation phase associated with splenomegaly and increased percentages (but not numbers) of Lyt-2+ and L3T4+ cells, followed by profound aplasia and abrogation of IL-2 production. Response to a B cell mitogen (LPS) is depressed, and cells from GVH mice exert a strong suppressive effect on the LPS and PHA responsiveness of normal cells. Suppression appears to be mediated by a radioresistant, nylon nonadherent, asialo GM1 negative cell expressing a low level of Thy-1 antigen. In contrast, protection correlates with progressive restoration of spleen cellularity and LPS responsiveness, with decreased but clearly detectable IL-2 production, and transient nonspecific suppressor activity. The immune status of protected mice resembles that of isografted controls. No correlation was found between mortality (or protection) and either PHA responsiveness, which remained depressed in all grafted mice throughout the observation period, or NK activity, which was strongly depressed in both GVH and protected mice. In conclusion, protection correlates with the disappearance of nonspecific suppressor cells and the restoration of cellularity and certain nonspecific immune functions. Donor immunization against host-specific non-H-2 antigens, which protects against mortality, also protects against GVH-associated immune deficiency.