A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans

Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.

The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells

The nematode Caenorhabditis elegans exhibits behavioral responses to many volatile odorants. Chemotaxis toward one such odorant, diacetyl (butanedione), requires the function of a seven-transmembrane receptor protein encoded by the odr-10 gene. To determine directly whether ODR-10 protein is an odorant receptor, it is necessary to express the protein in a heterologous system and show that it responds to diacetyl by activation of a G protein signaling pathway. Here we demonstrate that human cells expressing ODR-10 on their surfaces exhibit a transient elevation in intracellular Ca2+ levels after diacetyl application. Volatile compounds that differ from diacetyl only by the addition of a methyl group (2,3-pentanedione) or the absence of a keto group (butanone) are not ODR-10 agonists. Behavioral responses to these compounds are not dependent on odr-10 function, so ODR-10 specificity in human cells resembles in vivo specificity. The apparent affinity of ODR-10 for diacetyl observed in human cells is consistent with the diacetyl concentration ranges that allow efficient nematode chemotaxis. ODR-10 expressed in human cells also responds to two anionic compounds, pyruvate and citrate, which are metabolic precursors used for diacetyl production by certain bacterial species. Ca2+ elevation in response to ODR-10 activation is due to release from intracellular stores.

Perturbation of the T cell repertoire in rheumatoid arthritis

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) β-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR β-chain sequences of 1 in 2.4 × 107 CD4 T cells. In RA patients, the median TCR β-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P < 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P < 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.

Brain-derived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic abnormalities

Brain-derived neurotrophic factor (BDNF) has trophic effects on serotonergic (5-HT) neurons in the central nervous system. However, the role of endogenous BDNF in the development and function of these neurons has not been established in vivo because of the early postnatal lethality of BDNF null mice. In the present study, we use heterozygous BDNF+/− mice that have a normal life span and show that these animals develop enhanced intermale aggressiveness and hyperphagia accompanied by significant weight gain in early adulthood; these behavioral abnormalities are known to correlate with 5-HT dysfunction. Forebrain 5-HT levels and fiber density in BDNF+/− mice are normal at an early age but undergo premature age-associated decrements. However, young adult BDNF+/− mice show a blunted c-fos induction by the specific serotonin releaser-uptake inhibitor dexfenfluramine and alterations in the expression of several 5-HT receptors in the cortex, hippocampus, and hypothalamus. The heightened aggressiveness can be ameliorated by the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that endogenous BDNF is critical for the normal development and function of central 5-HT neurons and for the elaboration of behaviors that depend on these nerve cells. Therefore, BDNF+/− mice may provide a useful model to study human psychiatric disorders attributed to dysfunction of serotonergic neurons.

GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables positive and negative growth control of Chinese hamster ovary cells

We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27Kip1 in sense (p27Kip1S) and antisense (p27Kip1AS) orientation. Exclusive expression of p27Kip1S resulted in complete G1-phase-specific growth arrest, while expression of only p27Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27Kip1 failed to provide the expected G1-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.

Workshop on Schizophrenia

On November 29–30, 1995, the National Academy of Sciences and the Institute of Medicine brought together experts in schizophrenia and specialists in other areas of the biological sciences in a workshop aimed at promoting the application of the latest biological information to this clinical problem. The workshop paid particular attention to evidence of pathology in the brains of people with schizophrenia, and to the possibility that this reflects an abnormality in brain development that eventually leads to the appearance of symptoms. The participants were impressed with the complexity of the problem, and felt that multiple approaches would be required to understand this disease. They recommended that a major focus should be on the search for predisposing genes, but that there should be parallel research in many other areas.

Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification1

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.

Saving the Banks: The Political Economy of Bailouts. CES Papers - Open Forum #8, 2012

How much leeway did governments have in designing bank bailouts and deciding on the height of intervention during the 2007-2009 financial crisis? This paper analyzes comparatively what explains government responses to banking crises. Why does the type of intervention during financial crises vary to such a great extent across countries? By analyzing the variety of bailouts in Europe and North America, we will show that the strategies governments use to cope with the instability of financial markets does not depend on economic conditions alone. Rather, they take root in the institutional and political setting of each country and vary in particular according to the different types of business-government relations banks were able to entertain with public decision-makers. Still, “crony capitalism” accounts overstate the role of bank lobbying. With four case studies of the Irish, Danish, British and French bank bailout, we show that countries with close one-on-one relationships between policy-makers and bank management tended to develop unbalanced bailout packages, while countries where banks have strong interbank ties and collective negotiation capacity were able to develop solutions with a greater burden sharing from private institutions.

Lichtheimia Infection in a Lymphoma Patient: Case Report and a Brief Review of the Available Diagnostic Tools

We describe the case of a patient with a T-lymphoblastic lymphoma whose disseminated mucormycosis was diagnosed with delay, and we address the diagnostic and therapeutic decision-making process and review the diagnostic workup of patients with potential IFD. The diagnosis was delayed despite a suggestive radiological presentation of the patient's pulmonary lesion. The uncommon risk profile (T-lymphoblastic lymphoma, short neutropenic phases) wrongly led to a low level of suspicion. The diagnosis was also hampered by the lack of indirect markers for infections caused by Mucorales, the low sensitivity of both fungal culture and panfungal PCR, and the limited availability of species-specific PCR. A high level of suspicion of IFD is needed, and aggressive diagnostic procedures should be promptly initiated even in apparently low-risk patients with uncommon presentations. The extent of the analytical workup should be decided on a case-by-case base. Diagnostic tests such as the galactomannan and β-D-glucan test and/or PCR on biological material followed by sequencing should be chosen according to their availability and after evaluation of their specificity and sensitivity. In high-risk patients, preemptive therapy with a broad-spectrum mould-active antifungal agent should be started before definitive diagnostic findings become available.

Impfungen bei Immunsupprimierten. Mit Fokus auf HIV-positive Personen, Personen mit einer hämatologischen Stammzelltransplantation oder Organtransplantation und Personen mit funktionaler oder anatomischer Splenektomie

Personen mit einer HIV-Infektion, nach einer Organ- oder einer hämatologischen Stammzelltransplantation oder mit einer funktionalen oder anatomischen Asplenie sind gegenüber Infektionen anfälliger. Sie haben eine grössere Komplikationsrate und ein höheres Risiko für einen chronifizierten Verlauf. Impfungen wären eine ideale primäre Präventionsmassnahme, sind aber – durch dieselben Mechanismen des Immundefektes der zu schwereren Krankheitsverläufen führt – in ihrer Wirksamkeit vermindert. Die Impfungen sollen daher, wenn immer möglich, vor Beginn der Immunsuppression oder später zum Zeitpunkt der minimalsten Immunsuppression, durchgeführt werden. Trotzdem bleibt der Benefit von Impfungen bei immunsupprimierten Personen unbestritten, sofern die Indikationsstellung bezüglich Zeitpunkt und Dosierung (Dosismenge und -anzahl), die zu einem maximalen Ansprechen führt, beachtet wird. Lebendimpfungen sind wegen der Gefahr der unkontrollierten Vermehrung der Impfviren bei schwerer Immunsuppression kontraindiziert. Die Serologie soll unspezifischer gemessen werden, da schwer immunsupprimierte Personen im Falle einer relevanten Exposition durch passive Immunisierung mittels spezifischer oder unspezifischer intravenöser Immunglobuline geschützt werden können.

Travellers returning ill from the tropics – a descriptive retrospective study

nternational travel continues to increase in frequency. Health care providers need a wide understanding of the spectrum of travel related diseases and their management. This retrospective study analyses the demographic and clinical data of 360 travellers returning from the tropics presenting to an outpatient clinic at a tertiary hospital between 2003 - 2007. The aim of this study was to analyse the frequency of presenting symptoms and diseases in ill returning travellers and to correlate them to the areas visited and the duration and purpose of travel. The main symptoms during travel were diarrhoea (n = 200, 56 %) and fever (n = 124, 34 %). Travellers not visiting friends and relatives but with close contact to the local population were at more than two-fold increased risk of diarrhoea (Odds Ratio [OR] 2.5; 95 % confidence interval [CI] 1.1-6.0, p = 0.03) and fever (OR 2.4; 95 % CI 1.1-5.3; p = 0.02) compared to tourist travellers. Travellers visiting friends and relatives (VFR) were not at increased risk for diarrhoea (OR 0.6; 95 % CI 0.3-1.3; p = 0.17), or fever (OR 1.5; 95 % CI 0.7-3.4; p = 0.28). Thirty-two percent of all travellers (n = 115) were diagnosed with a specific pathogen. Malaria (6 %), giardiasis (6 %) and amebiasis (4 %) were the most frequently detected pathogens. The odds of malaria as a cause of the presenting illness was lower among travellers reporting pre-travel advice. Specific antimicrobial treatment was required in around one third of the patients.