Structural Organization of Perisomatic Inhibition in the Mouse Medial Prefrontal Cortex.

Perisomatic inhibition profoundly controls neural function. However, the structural organization of inhibitory circuits giving rise to the perisomatic inhibition in the higher-order cortices is not completely known. Here, we performed a comprehensive analysis of those GABAergic cells in the medial prefrontal cortex (mPFC) that provide inputs onto the somata and proximal dendrites of pyramidal neurons. Our results show that most GABAergic axonal varicosities contacting the perisomatic region of superficial (layer 2/3) and deep (layer 5) pyramidal cells express parvalbumin (PV) or cannabinoid receptor type 1 (CB1). Further, we found that the ratio of PV/CB1 GABAergic inputs is larger on the somatic membrane surface of pyramidal tract neurons in comparison with those projecting to the contralateral hemisphere. Our morphologic analysis of in vitro labeled PV+ basket cells (PVBC) and CCK/CB1+ basket cells (CCKBC) revealed differences in many features. PVBC dendrites and axons arborized preferentially within the layer where their soma was located. In contrast, the axons of CCKBCs expanded throughout layers, although their dendrites were found preferentially either in superficial or deep layers. Finally, using anterograde trans-synaptic tracing we observed that PVBCs are preferentially innervated by thalamic and basal amygdala afferents in layers 5a and 5b, respectively. Thus, our results suggest that PVBCs can control the local circuit operation in a layer-specific manner via their characteristic arborization, whereas CCKBCs rather provide cross-layer inhibition in the mPFC.SIGNIFICANCE STATEMENT Inhibitory cells in cortical circuits are crucial for the precise control of local network activity. Nevertheless, in higher-order cortical areas that are involved in cognitive functions like decision-making, working memory, and cognitive flexibility, the structural organization of inhibitory cell circuits is not completely understood. In this study we show that perisomatic inhibitory control of excitatory cells in the medial prefrontal cortex is performed by two types of basket cells endowed with different morphologic properties that provide inhibitory inputs with distinct layer specificity on cells projecting to disparate areas. Revealing this difference in innervation strategy of the two basket cell types is a key step toward understanding how they fulfill their distinct roles in cortical network operations.

Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC.

Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be "silent" in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated.

Molecular insights into the role of heme in the transcriptional regulatory system AppA/PpsR.

Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant Kd for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.

Total Number and Ratio of GABAergic Neuron Types in the Mouse Lateral and Basal Amygdala.

GABAergic neurons are key circuit elements in cortical networks. Despite growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types has been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22%) is significantly higher than in the LA (16%) in both male and female mice. No difference was observed between the right and left hemispheres in either sex. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5%-6%), basket cells expressing parvalbumin (17%-20%) or cholecystokinin (7%-9%), dendrite-targeting inhibitory cells expressing somatostatin (10%-16%), NPY-containing neurogliaform cells (14%-15%), VIP and/or calretinin-expressing interneuron-selective interneurons (29%-38%), and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (5.5%-8%). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathologic conditions, and to model functioning amygdalar networks in health and disease.SIGNIFICANCE STATEMENT GABAergic cells in cortical structures, as in the lateral and basal nucleus of the amygdala, have a determinant role in controlling circuit operation. In this study, we provide the first estimate for the total number of inhibitory cells in these two amygdalar nuclei. In addition, our study is the first to define the ratio of the major GABAergic cell types present in these cortical networks. Taking into account that hyperexcitability in the amygdala, arising from the imbalance between excitation and inhibition typifies many altered brain functions, including anxiety, post-traumatic stress disorder, schizophrenia, and autism, uncovering the number and ratio of distinct amygdalar inhibitory cell types offers a solid base for comparing the changes in inhibition in pathologic brain states.

The C-terminal tail extension of myosin 16 acts as a molten globule, including intrinsically disordered regions, and interacts with the N-terminal ankyrin.

The lesser-known unconventional myosin 16 protein is essential in proper neuronal functioning and has been implicated in cell cycle regulation. Its longer Myo16b isoform contains a C-terminal tail extension (Myo16Tail), which has been shown to play a role in the neuronal phosphoinositide 3-kinase signaling pathway. Myo16Tail mediates the actin cytoskeleton remodeling, downregulates the actin dynamics at the postsynaptic site of dendritic spines, and is involved in the organization of the presynaptic axon terminals. However, the functional and structural features of this C-terminal tail extension are not well known. Here, we report the purification and biophysical characterization of the Myo16Tail by bioinformatics, fluorescence spectroscopy, and CD. Our results revealed that the Myo16Tail is functionally active and interacts with the N-terminal ankyrin domain of myosin 16, suggesting an intramolecular binding between the C and N termini of Myo16 as an autoregulatory mechanism involving backfolding of the motor domain. In addition, the Myo16Tail possesses high structural flexibility and a solvent-exposed hydrophobic core, indicating the largely unstructured, intrinsically disordered nature of this protein region. Some secondary structure elements were also observed, indicating that the Myo16Tail likely adopts a molten globule-like structure. These structural features imply that the Myo16Tail may function as a flexible display site particularly relevant in post-translational modifications, regulatory functions such as backfolding, and phosphoinositide 3-kinase signaling.

Identification of the vibrational marker of tyrosine cation radical using ultrafast transient infrared spectroscopy of flavoprotein systems.

Tryptophan and tyrosine radical intermediates play crucial roles in many biological charge transfer processes. Particularly in flavoprotein photochemistry, short-lived reaction intermediates can be studied by the complementary techniques of ultrafast visible and infrared spectroscopy. The spectral properties of tryptophan radical are well established, and the formation of neutral tyrosine radicals has been observed in many biological processes. However, only recently, the formation of a cation tyrosine radical was observed by transient visible spectroscopy in a few systems. Here, we assigned the infrared vibrational markers of the cationic and neutral tyrosine radical at 1483 and 1502 cm-1 (in deuterated buffer), respectively, in a variant of the bacterial methyl transferase TrmFO, and in the native glucose oxidase. In addition, we studied a mutant of AppABLUF blue-light sensor domain from Rhodobacter sphaeroides in which only a direct formation of the neutral radical was observed. Our studies highlight the exquisite sensitivity of transient infrared spectroscopy to low concentrations of specific radicals.

Hepcidin and its potential clinical utility.

A number of pathophysiological conditions are related to iron metabolism disturbances. Some of them are well known, others are newly discovered or special. Hepcidin is a newly identified iron metabolism regulating hormone, which could be a promising biomarker for many disorders. In this review, we provide background information about mammalian iron metabolism, cellular iron trafficking, and the regulation of expression of hepcidin. Beside these molecular biological processes, we summarize the methods that have been used to determine blood and urine hepcidin levels and present those pathological conditions (cancer, inflammation, neurological disorders) when hepcidin measurement may have clinical relevance.

Prohepcidin binds to the HAMP promoter and autoregulates its own expression.

Hepcidin is the major regulatory peptide hormone of iron metabolism, encoded by the HAMP (hepcidin antimicrobial peptide) gene. Hepcidin is expressed mainly in hepatocytes, but is also found in the blood in both a mature and prohormone form. Although, the function of mature hepcidin and the regulation of the HAMP gene have been extensively studied, the intracellular localization and the fate of prohepcidin remains controversial. In the present study, we propose a novel role for prohepcidin in the regulation of its own transcription. Using indirect immunofluorescence and mCherry tagging, a portion of prohepcidin was detected in the nucleus of hepatocytes. Prohepcidin was found to specifically bind to the STAT3 (signal transducer and activator of transcription 3) site in the promoter of HAMP. Overexpression of prohepcidin in WRL68 cells decreased HAMP promoter activity, whereas decreasing the amount of prohepcidin caused increased promoter activity measured by a luciferase reporter-gene assay. Moreover, overexpression of the known prohepcidin-binding partner, α-1 antitrypsin caused increased HAMP promoter activity, suggesting that only the non-α-1 antitrypsin-bound prohepcidin affects the expression of its own gene. The results of the present study indicate that prohepcidin can bind to and transcriptionally regulate the expression of HAMP, suggesting a novel autoregulatory pathway of hepcidin gene expression in hepatocytes.

Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State.

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent 'on' state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.

Fear learning and aversive stimuli differentially change excitatory synaptic transmission in perisomatic inhibitory cells of the basal amygdala.

Inhibitory circuits in the basal amygdala (BA) have been shown to play a crucial role in associative fear learning. How the excitatory synaptic inputs received by BA GABAergic interneurons are influenced by memory formation, a network parameter that may contribute to learning processes, is still largely unknown. Here, we investigated the features of excitatory synaptic transmission received by the three types of perisomatic inhibitory interneurons upon cue-dependent fear conditioning and aversive stimulus and tone presentations without association. Acute slices were prepared from transgenic mice: one group received tone presentation only (conditioned stimulus, CS group), the second group was challenged by mild electrical shocks unpaired with the CS (unsigned unconditioned stimulus, unsigned US group) and the third group was presented with the CS paired with the US (signed US group). We found that excitatory synaptic inputs (miniature excitatory postsynaptic currents, mEPSCs) recorded in distinct interneuron types in the BA showed plastic changes with different patterns. Parvalbumin (PV) basket cells in the unsigned US and signed US group received mEPSCs with reduced amplitude and rate in comparison to the only CS group. Coupling the US and CS in the signed US group caused a slight increase in the amplitude of the events in comparison to the unsigned US group, where the association of CS and US does not take place. Excitatory synaptic inputs onto cholecystokinin (CCK) basket cells showed a markedly different change from PV basket cells in these behavioral paradigms: only the decay time was significantly faster in the unsigned US group compared to the only CS group, whereas the amplitude of mEPSCs increased in the signed US group compared to the only CS group. Excitatory synaptic inputs received by PV axo-axonic cells showed the least difference in the three behavioral paradigm: the only significant change was that the rate of mEPSCs increased in the signed US group when compared to the only CS group. These results collectively show that associative learning and aversive stimuli unpaired with CS cause different changes in excitatory synaptic transmission in BA perisomatic interneuron types, supporting the hypothesis that they play distinct roles in the BA network operations upon pain information processing and fear memory formation.

Chemical-neuroanatomical organization of peripheral sensory-efferent systems in the pond snail (Lymnaea stagnalis).

Perception and processing of chemical cues are crucial for aquatic gastropods, for proper elaboration of adaptive behavior. The pond snail, Lymnaea stagnalis, is a model species of invertebrate neurobiology, in which peripheral sensory neurons with different morphology and transmitter content have partly been described, but we have little knowledge regarding their functional morphological organization, including their possible peripheral intercellular connections and networks. Therefore the aim of our study was to characterize the sensory system of the tentacles and the lip, as primary sensory regions, and the anterior foot of Lymnaea with special attention to the transmitter content of the sensory neurons, and their relationship to extrinsic elements of the central nervous system. Numerous bipolar sensory cells were demonstrated in the epithelial layer of the peripheral organs, displaying immunoreactivity to antibodies raised against tyrosine hydroxylase, histamine, glutamate and two molluscan type oligopeptides, FMRFamide and Mytilus inhibitory peptide. A subepithelial plexus was formed by extrinsic serotonin and FMRFamide immunoreactive fibers, whereas in deeper regions axon processess of different origin with various immunoreactivities formed networks, too. HPLC-MS assay confirmed the presence of the low molecular weight signal molecules in the three examined areas. Following double-labeling immunohistochemistry, close arrangements were observed, formed by sensory neurons and extrinsic serotonergic (and FMRFamidergic) fibers at axo-dendritic, axo-somatic and axo-axonic levels. Our results suggest the involvement of a much wider repertoire of signal molecules in peripheral sensory processes of Lymnaea, which can locally be modified by central input, hence influencing directly the responses to environmental cues.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy.

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

The C19S Substitution Enhances the Stability of Hepcidin While Conserving Its Biological Activity.

Hepcidin, the key hormone of iron homeostasis is responsible for lowering the serum iron level through its interaction with iron exporter ferroportin. Thus, hepcidin agonists provide a promising opportunity in the treatment of iron disorders caused by lacking or decreased hepcidin expression. We investigated the importance of each of the eight highly conserved cysteines for the biological activity of hepcidin. Eight cysteine mutants were created with site directed mutagenesis. The binding ability of these hepcidin mutants to the hepcidin receptor ferroportin was determined using bacterial two-hybrid system and WRL68 human hepatic cells. The biological activity of hepcidin mutants was determined by western blot analysis of ferroportin internalization and ferroportin ubiquitination. To investigate the effect of mutant hepcidins on the iron metabolism of the WRL68 cells, total intracellular iron content was measured with a colorimetric assay. The stability of M6 hepcidin mutant was determined using ELISA technique. Our data revealed that serine substitution of the sixth cysteine (M6) yielded a biologically active but significantly more stable peptide than the original hormone. This result may provide a promising hepcidin agonist worth testing in animal models.

[Molecular detection of adenovirus type-8 epidemic keratoconjunctivitis in Hungary]. / Adenovírus 8-as típusa okozta járványos keratoconjunctivitis molekuláris diagnosztikája.

INTRODUCTION: Both infectious and non-infectious forms of acute conjunctivitis are known. Viruses, especially different types of adenoviruses are the etiological agents of infectious epidemic conjunctivitis (conjunctivitis epidemica). AIMS: The author's aims were to describe an outbreak of keratoconjunctivitis and to detect the viral agent by molecular methods in Hungary. MATERIALS AND METHODS: Classical epidemiological methods were used for investigation. Polymerase chain reaction (PCR) followed by sequencing were used for the detection of adenoviral hexon region from freshly collected conjunctival swabs. RESULTS: Between 9 October and 18 December 2006, a total of 60 patients became ill with keratoconjunctivitis in 7 settlements in Southwest Hungary. Mean age was 51,2 years. Conjunctivitis (100%), lacrimation (94%), foreign body sensation (83%), and dim vision (76%) were the main clinical symptoms. Both eyes were affected in half of the cases. Direct contact was the main transmission route including nosocomial spread associated with ophthalmology practices. Five (62.5%) of 8 conjunctival swabs were PCR-positive for adenovirus type 8 (HAdV8/Baranya/2006/HUN; EF210714) which was genetically identical to adenovirus strain detected in Austria in 2004 (DQ149614). CONCLUSIONS: The outbreak of keratoconjunctivitis was partially associated with nosocomial infection caused by type 8 adenovirus. Both the recognition of the clinical illness, laboratory diagnosis and public health measures are necessary for the prevention of keratoconjunctivitis infection and epidemic.

[Hepatitis A outbreak in Transdanubia (Hungary): molecular connections and epidemiological conclusions - 2006]. / Hepatitis A-járvány a Dél-Dunántúlon: molekuláris összefüggések, epidemiológiai következtetések - 2006.

INTRODUCTION: Hepatitis A virus (HAV) is one of the most important cause of fecally transmitted acute infectious hepatitis worldwide. In Hungary, beside the sporadic HAV infections, outbreaks also occur, particularly in Northeast part of the country where the subgenotype IA is endemic. The reported number of HAV cases was less than 10 per year in Southwest Hungary. AIMS: A part of the European HAV surveillance, the author's aims were to follow and to analyze the outbreak of hepatitis A in Transdanubia (Southwest Hungary) in 2006 by molecular epidemiological methods. MATERIALS AND METHODS: Sera samples from symptomatic patients were tested prospectively by enzyme-immunoassay (EIA) and reverse transcription-polymerase chain reaction (RT-PCR) followed by sequence- and phylogenetic analysis. RESULTS: Between June and December 2006, a total of 115 serum samples were positive for HAV IgM antibody in Southwest Hungary. Thirty (76.9%) of the 39 samples were RT-PCR-positive and contain genetically identical subgenotype IB hepatitis A virus (HAV/Transdanubia/2006/HUN) which has 100% nucleotide identity to strain IT-MAR-02 from Italy in 2002. Until now, approximately 1200-1300 persons have been infected with the probably imported strain. The average age of patients was 18 years (years 1 to 80). In this study, detailed clinical and epidemiological data of the outbreak are presented. CONCLUSIONS: Only a prospective molecular epidemiological study could verify the connection between the first sporadic HAV cases in June, the outbreak in August and the endemic spread of the virus since September in Southwest Hungary. This epidemic calls attention to how a hepatitis A outbreak can develop anywhere and at any time in Hungary and to the importance of active HAV immunization in the primary prevention.

Blood-brain barrier changes during compensated and decompensated hemorrhagic shock.

Dysfunction of the blood-brain barrier (BBB) can be associated with a large number of central nervous system and systemic disorders. The aim of the present study was to determine BBB changes during different phases of hemorrhagic shock. The experiments were carried out on male Wistar rats anaesthetized with urethane. To produce compensated or decompensated hemorrhagic shock, mean arterial pressure was decreased from the normotensive control values to 40 mmHg by a standardized method of blood withdrawal from the femoral artery. Cerebral blood flow changes were followed by laser-Doppler flowmetry, and arterial blood gas values were monitored over the whole procedure. Cortical blood flow was significantly reduced in compensated and in decompensated hemorrhagic shock compared with the normotensive rats. As the shock shifted to the decompensated phase, the blood flow reduction was more pronounced. BBB permeability studies using sodium fluorescein (molecular weight of 376) and Evan's Blue albumin (molecular weight of 67,000) have revealed a significant increase of the BBB permeability for sodium fluorescein in the decompensated stage of hemorrhagic shock. Western blot analysis of brain capillaries showed that the expression of the transmembrane tight junction protein occludin was reduced in response to hemorrhagic shock, and the decrease of occludin was more pronounced in the decompensated phase. A similar expression pattern was shown by the transmembrane adherens junction protein cadherin as well. Our results suggest that the decompensated phase of hemorrhagic shock is associated with disturbances of the BBB, which may be explained by the dysfunction of interendothelial junctions caused by decreased occludin and cadherin levels.

Pet127 governs a 5' -> 3'-exonuclease important in maturation of apocytochrome b mRNA in Saccharomyces cerevisiae.

The details of mRNA maturation in Saccharomyces mitochondria are not well understood. All seven mRNAs are transcribed as part of multigenic units. The mRNAs are processed at a common 3'-dodecamer sequence, but the 5'-ends have seven different sequences. To investigate whether apocytochrome b (COB) mRNA is processed at the 5'-end from a longer precursor by an endonuclease or an exonuclease, a 64-nucleotide sequence, which is required for the protection of COB mRNA by the Cbp1 protein and is found at the 5'-end of the processed COB mRNA, was duplicated in tandem. The wild-type 64-nucleotide element functioned in either the upstream or downstream position when paired with a mutant element. In the tandem wild-type strain, the 5'-end of the mRNA was at the 5'-end of the upstream unit, demonstrating that the mRNA is processed by an exonuclease. Accumulation of precursor COB RNA in single and double element strains with a deletion of PET127 demonstrated that the encoded protein governs the 5'-exonuclease responsible for processing the precursor to the mature form.

Co-circulation of genotype IA and new variant IB hepatitis A virus in outbreaks of acute hepatitis in Hungary--2003/2004.

Hepatitis A virus (HAV) is one of the most important causes of acute infectious hepatitis worldwide. In Hungary, the reported number of HAV infections has been decreasing in the last four decades, nevertheless, still, each year 500-800 new cases and multiple outbreaks occur, particularly in the northeast region of Hungary. In Hungary, serology is used routinely to establish the diagnosis of HAV infection without genetic analysis of HAV strains for molecular epidemiology. In this study, serum samples collected from symptomatic patients were tested by enzyme-immunoassay (anti-HAV-IgM ELISA) to establish the cause of three acute hepatitis A outbreaks (outbreak 1--from low prevalence region in Southwest Hungary in 2003 and outbreaks 2 and 3 from the endemic region in Northeast Hungary in 2004). Outbreak strains were characterized by reverse transcription-polymerase chain reaction (RT-PCR) amplification of a 360 bp viral VP1/2A region, amplicon sequencing and phylogenetic analysis. Four, seven, and three sera from outbreaks 1, 2, and 3, respectively, were investigated by RT-PCR for HAV genome and HAV RNA was detected in 4 (100%), 4 (57%), and 2 (67%) samples. All strains belonged to genotype I HAV. Outbreak 1 was caused by the new variant subtype IB and outbreaks 2 and 3 caused by genetically identical subtype IA strains. The Hungarian IA and IB hepatitis A viruses had the highest nucleotide identity, 98.4% and 99.0%, to IT-SCH-00 and IT-MAR-02 strains, respectively, detected in year 2000 and 2002 in Italy. Endemic subtype IA and probably imported new variant subtype IB HAV viruses was detected in outbreaks of hepatitis in Hungary that are closely related genetically to HAV strains in Italy.

Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria.

Mitochondria perform a central function in the biogenesis of cellular iron-sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP-binding cassette (ABC) protein Rli1p carries N-terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p.

Maturation of cytosolic iron-sulfur proteins requires glutathione.

Glutathione is the major protective agent against oxidative stress in Saccharomyces cerevisiae. Deletion of the GSH1 gene (strain Deltagsh1) encoding the enzyme that catalyzes the first step of glutathione biosynthesis leads to growth arrest, which can be relieved by either glutathione or reducing agents such as dithiothreitol. Because defects in the biosynthesis of cellular iron-sulfur (Fe/S) proteins are associated with increases in glutathione levels, we examined the consequences of glutathione depletion on this essential process. No significant defects were detected in the amounts, activities, and maturation of mitochondrial Fe/S proteins in glutathione-depleted Deltagsh1 cells. On the contrary, the maturation of extra-mitochondrial Fe/S proteins was decreased substantially. The defect was rectified neither by addition of dithiothreitol nor under anaerobic conditions excluding oxidative damage of Fe/S clusters. A double mutant in GSH1 and ATM1 encoding a mitochondrial ATP binding cassette (ABC) transporter involved in cytosolic Fe/S protein maturation is nonviable even in the presence of dithiothreitol. Similar to atm1 and other mutants defective in cytosolic Fe/S protein maturation, mitochondria from glutathione-depleted Deltagsh1 cells accumulated high amounts of iron. Together, our data demonstrate that glutathione, in addition to its protective role against oxidative damage, performs a novel and specific function in the maturation of cytosolic Fe/S proteins.