BACKGROUND

Rotavirus is the most common causes of severe, acute diarrhea during childhood and is an important cause of morbidity and mortality in developing countries. We established active hospital-based surveillance of childhood diarrhea to assess the scope of severe rotavirus disease in Iran.

METHODS

From May 2006 through April 2007, prospective surveillance of rotavirus diarrhea among children aged <5 years was conducted in 5 sentinel hospitals in Iran. Stool samples were tested for rotavirus using a commercially available enzyme immunoassay, and rotavirus-positive samples were genotyped using reverse-transcriptase polymerase chain reaction.

RESULTS

Of 2198 children admitted to the hospital for acute gastroenteritis, 1298 (59.1%) had stool samples test positive for rotavirus by enzyme immunoassay. Of the rotavirus episodes, 85% occurred during the first 2 years of life, with the peak prevalence of severe rotavirus disease occurring from September through January. Among the 110 rotavirus-positive samples that were genotyped, G4P[8] was the most commonly detected rotavirus genotype (30.9% of strains). Other commonly detected genotypes included P[8] with G nontypeable (21.8%), G4 with P nontypeable (13.6%), G1[P8] (10.9%), and G2[P4] (5.5%).

CONCLUSIONS

Rotavirus is the most common cause of severe diarrhea in Iran, which indicates that safe and effective rotavirus vaccination in Iran is a public health priority.

A spatial and temporal analysis of auditory evoked potentials within the brain stem were performed in cats to determine the areas of the brain stem having large amplitude voltage fields, corresponding in latency to each of the components of the scalp-recorded auditory brain stem response (ABR). On the basis of this criterion, the first few components (occurring within 2 msec post-stimulus) were attributed to activity in a single structure, the eighth nerve. In contrast, each of the other components was correlated with large amplitude fields in at least two sites within the brain stem auditory pathways. The findings demonstrate a complex spatial and temporal distribution of electrical events within the auditory brain stem pathways, which preclude any simple one-to-one relationship between a given anatomical site and a particular component of the ABR. The possibility that the determination of the generators might be influenced by filtering of the evoked potentials was also examined. High-pass filtering of the evoked potentials resulted in a modification of the defined generators for only one of the components studied (P4). Filtering had little effect on the components of the scalp-recorded ABR.

Integrated hepatitis B virus (HBV) DNA is found in hepatocellular carcinomas which develop in HBV carriers. Presented here are the results of analyses of four integrants that show chromosomal rearrangements associated with the integrated HBV DNA. Two clones (p4 and C15) were found to have large inverted repeating structures, each consisting of HBV genome along with flanking cellular sequences. The structure must have arisen by duplication of the primary integrant, including the flanking cellular DNA, followed by recombination within the viral DNA. One of the two viral arms in each clone joins to the other viral arm at the "cohesive end region." Two clones (DA2-2 and DA2-6) were found to have integrated HBV sequences, each flanked by cellular DNAs from different chromosomes (chromosome X joined to 17 and chromosome 5 joined to 9). They must be the products of cellular DNA translocations using the integrated HBV DNA as the switch point. The viral DNA in each clone is a continuous stretch of a single virus genome with one end in the cohesive end region. These complex structures seem to have been produced by activation of the cohesive end of an integrant viral genome, followed by its recombination with another chromosomal DNA.

Synaptogenesis was studied in the rat suprachiasmatic nucleus (SCN) using quantitative ultrastructural analysis and synapsin I immunohistochemistry. SCN neurons are formed between embryonic days 13 and 17 (E13-E17), and the SCN is a distinct hypothalamic nucleus by E18. At E19 the nucleus is very immature and there are few synapses. Synaptogenesis proceeds slowly until P4 but increases rapidly between P4 and P10. At P10 the number of synapses per unit area is the same as in the adult SCN and all synaptic types present in the adult are evident. However, the SCN continues to increase in volume to the adult with approximately 30% of total synaptic number added between P10 and a young adult age. The appearance of synapsin I immunoreactivity correlates very precisely with the development of synapses in the SCN as shown by ultrastructural analysis between E19 and P6. The pattern of appearance of synapsin I immunoreactivity demonstrates that synaptogenesis in the SCN is significantly delayed in comparison to adjacent hypothalamic nuclei. Synapsin I immunohistochemistry is a reliable marker of synapse formation in the developing SCN. A correlation of these anatomical data with prior functional studies suggests that SCN neurons are born as individual circadian oscillators that undergo a rapid development in the first 10 days after birth to form a functional neural network subserving circadian rhythm generation and regulation.

Mutations in the maternal-effect sterile gene mes-1 cause the offspring of homozygous mutant mothers to develop into sterile adults. Lineage analysis revealed that mutant offspring are sterile because they fail to form primordial germ cells during embryogenesis. In wild-type embryos, the primordial germ cell P4 is generated via a series of four unequal stem-cell divisions of the zygote. mes-1 embryos display a premature and progressive loss of polarity in these divisions: P0 and P1 undergo apparently normal unequal divisions and cytoplasmic partitioning, but P2 (in some embryos) and P3 (in most embryos) display defects in cleavage asymmetry and fail to partition lineage-specific components to only one daughter cell. As an apparent consequence of these defects, P4 is transformed into a muscle precursor, like its somatic sister cell D, and generates up to 20 body muscle cells instead of germ cells. Our results show that the wild-type mes-1 gene participates in promoting unequal germ-line divisions and asymmetric partitioning events and thus the determination of cell fate in early C. elegans embryos.

The pharmacological activity of diadenosine polyphosphates was investigated at three recombinant P2X receptors (rat P2X1, rat P2X3, rat P2X4) expressed in Xenopus oocytes and studied under voltage-clamp conditions. For the rat P2X1 receptor, only P1,P6-diadenosine hexaphosphate (Ap6A) was a full agonist yet 2-3 folds less potent than ATP. At rat P2X3, P1,p4-diadenosine tetraphosphate (Ap4A), P1,P5-diadenosine pentaphosphate (Ap5A) and Ap6A were full agonists and more potent than ATP. Ap4A alone was equipotent with ATP at rat P2X4, but only as a partial agonist. Compared to known data for rat P2X2 and human P2X1 receptors, our findings contrast with rat P2X2 where only Ap4A is a full agonist although four folds less potent than ATP. At rat and human orthologues of P2X1, Ap5A was a partial agonist with similar potency. These data provide a useful basis for selective agonists of P2X receptor subunits.

The vir genes of Agrobacterium tumefaciens tumor-inducing (Ti) plasmids direct the transfer of oncogenic portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells (T-DNA) into plant cells and are coordinately induced by plant-released phenolic chemical signals. We have used DNA microarrays, representing all genes of the octopine- and nopaline-type Ti plasmids, to identify all Ti-plasmid-encoded genes in the vir regulons of both plasmids. Acetosyringone (AS) induced the expression of all known members of the vir regulons, as well as a small number of additional genes. Unexpectedly, AS also caused a modest induction of virtually every Ti plasmid gene. This suggested that the copy number of the Ti plasmid might increase in response to AS, a hypothesis confirmed by DNA dot blotting. VirA and VirG were the only Vir proteins required for this copy number increase. Promoter resections and primer extension analysis of the repABC promoter region showed that expression of the promoter closest to repA (promoter P4) was induced by AS. We also identified a sequence resembling a consensus VirG-binding motif approximately 70 nucleotides upstream from the P4 transcription start site. Mutating this sequence blocked the AS-induced copy number increase of a RepABC-dependent miniplasmid, indicating that phospho-VirG increases copy number solely by enhancing repABC expression.

By using the minute virus of mice, we have shown that in vivo and in vitro RNA polymerase II pauses or prematurely terminates transcription at a specific location 142-147 nucleotides downstream from the P4 promoter. The attenuated RNA was found and mapped in vivo in A9 cell late after infection in both the nuclear and cytoplasmic fractions, and the terminal nucleotide was shown to have a 3' OH group. The 3' end of the attenuated RNA is capable of forming a hairpin structure that is followed by a stretch of uridines. To distinguish whether the attenuated RNA is formed as a result of processing, pausing, or termination and to dissect structural elements, factors, or mechanisms that are involved in its formation, we used in vitro systems: isolated nuclei and cell-free extracts from HeLa cells. The results of the in vitro studies show that the attenuated RNA is a result of pausing or termination and not processing. Additionally, a salt-soluble factor and RNA secondary structure were implicated in the process of termination.

Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.

SENPs [Sentrin/SUMO (small ubiquitin-related modifier)-specific proteases] include proteases that activate the precursors of SUMOs, or deconjugate SUMOs attached to target proteins. SENPs are usually assayed on protein substrates, and for the first time we demonstrate that synthetic substrates can be convenient tools in determining activity and specificity of these proteases. We synthesized a group of short synthetic peptide fluorogenic molecules based on the cleavage site within SUMOs. We demonstrate the activity of human SENP1, 2, 5, 6, 7 and 8 on these substrates. A parallel positional scanning approach using a fluorogenic tetrapeptide library established preferences of SENPs in the P3 and P4 positions that allowed us to design optimal peptidyl reporter substrates. We show that the specificity of SENP1, 2, 5 and 8 on the optimal peptidyl substrates matches their natural protein substrates, and that the presence of the SUMO domain enhances catalysis by 2-3 orders of magnitude. We also show that SENP6 and 7 have an unexpected specificity that distinguishes them from other members of the family, implying that, in contrast to previous predictions, their natural substrate(s) may not be SUMO conjugates.

The Hemophilus influenzae Hap adhesin is an autotransporter protein that undergoes an autoproteolytic cleavage event resulting in extracellular release of the adhesin domain (Hap(s)) from the membrane-associated translocator domain (Hap(beta)). Hap autoproteolysis is mediated by Ser(243) and occurs at LN1036-7 and to a lesser extent at more COOH-terminal alternate sites. In the present study, we sought to further define the mechanism of Hap autoproteolysis. Site-directed mutagenesis of residues His(98) and Asp(140) identified a catalytic triad conserved among a subfamily of autotransporters and reminiscent of the SA (chymotrypsin) clan of serine proteases. Amino-terminal amino acid sequencing of histidine-tagged Hap(beta) species and site-directed mutagenesis established that autoproteolysis occurs at LT1046-7, FA1077-8, and FS1067-8, revealing a consensus target sequence for cleavage that consists of ((Q/R)(A/S)X(L/F)) at the P4 through P1 positions. Examination of a recombinant strain co-expressing a Hap derivative lacking all cleavage sites (HapDelta1036-99) and a Hap derivative lacking proteolytic activity (HapS243A) demonstrated that autoproteolysis occurs by an intermolecular mechanism. Kinetic analysis of Hap autoproteolysis in bacteria expressing Hap under control of an inducible promoter demonstrated that autoproteolysis increases as the density of Hap precursor in the outer membrane increases, confirming intermolecular cleavage and suggesting a novel mechanism for regulation of bacterial adherence and microcolony formation.

Bacteriophage phi 6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (l, m, s). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4 and P7. Expression of the cDNA copy of the L segment in Escherichia coli leads to the formation of empty procapsid particles. These particles are able to package the plus-sense single-stranded RNA (ssRNA)s of each genome segment in vitro. We have used this in vitro system for a detailed study of phi 6 RNA packaging. The reaction conditions for RNA packaging were optimized using a RNase protection assay. The RNA packaging reaction is dependent on divalent cations (either Mg2+ or Mn2+) and requires a nucleoside triphosphate (NTP) as an energy source. Any one of the rNTPs, dNTPs or ddNTPs can support the RNA packaging. Purine nucleotides support packaging better than pyrimidine nucleotides, GTP being preferred to ATP. The plus-sense ssRNA of each the three genome segments can be packaged independently into the procapsid. However, when two or three segments are packaged simultaneously, regulatory effects modulating the packaging efficiency can be detected between the segments. The packaging of the s and m segments is more efficient when they are packaged alone, compared to a situation in which they are packaged with the other segments. In contrast, the packaging of the l segment is very inefficient alone, but is enhanced when packaged together with the m segment. We propose that each segment has a preferred high-affinity binding site in the procapsid particle and packaging of the m segment creates the high-affinity binding site for the l segment. If any of the segments is missing from the packaging reaction the other segments can occupy its binding site.

Ovarian steroids increase hypothalamic-pituitary-adrenal (HPA) axis activity and sensitize the hypothalamic-pituitary-ovarian (HPO) axis to stress-induced inhibition. The present study investigated the effect of ovarian steroids on CRH and arginine vasopressin (AVP) messenger RNA (mRNA) levels in the rhesus monkey hypothalamus, as both neuropeptides have been shown to stimulate the HPA axis and inhibit the HPO axis in this species. This was accomplished by measuring CRH and AVP mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization histochemistry. Menstrual cycles were simulated in ovariectomized (OVX) rhesus monkeys by sequential addition and removal of SILASTIC brand (Dow Corning Corp.) tubing containing either 17beta-estradiol (E2) or progesterone (P4). On the morning of day 11 of the simulated follicular phase (E2 alone) or day 21 of the luteal phase (E2 + P4), animals were anesthetized, and the brains were perfused with paraformaldehyde via the carotid artery. Coronal sections (30 microm) were cut, and mRNA for CRH and AVP in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) were semiquantified by in situ hybridization. CRH mRNA in the PVN of E2-replaced OVX animals (n = 7) was 2-fold greater than that in untreated OVX controls (n = 4), whereas CRH mRNA after E2 + P4 (n = 4) was no different from that in controls (optical density + SEM, 0.38 +/- 0.06, 0.13 +/- 0.08, and 0.14 +/- 0.09 for OVX + E2, OVX + E2 + P4, and OVX, respectively; P = 0.02). CRH in the SON was undetectable. In contrast to CRH, AVP mRNA in the PVN and the SON was similar in the three treatment groups. We conclude that E2 and E2 + P4 replacement to OVX monkeys exert different effects on CRH and AVP gene expression, as estrogen stimulation of CRH mRNA in the PVN was abrogated by progesterone, whereas no effect of ovarian steroids on AVP mRNA in either the PVN or SON was observed. We postulate that ovarian steroid regulation of CRH synthesis and release may in part explain the central nervous system mechanisms by which ovarian steroids affect the HPA and HPO axes during basal and stress conditions.

Asymmetrical distribution of phospholipids is generally observed in the eukaryotic plasma membrane. Maintenance and changes of this phospholipid asymmetry are regulated by ATP-driven phospholipid translocases. Accumulating evidence indicates that type 4 P-type ATPases (P4-ATPases, also called flippases) translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the plasma membrane and internal membranes. Among P-type ATPases, P4-ATPases are unique in that they are associated with a conserved membrane protein of the Cdc50 family as a non-catalytic subunit. Recent studies indicate that flippases are involved in various cellular functions, including transport vesicle formation and cell polarity. In this review, we will focus on the functional aspect of phospholipid flippases.

The early stages in the formation of Purkinje cell dendritic arbors have been analyzed using the horseradish peroxidase (HRP) 'in vitro' axonal tracing method, from embryonic day 19 (E19) to postnatal day 6 (P6). These stages comprise the transition from the bipolar Purkinje cell, at the end of its migration, to the phase of stellate cell with disoriented dendrites. Postmigratory Purkinje cells in the cortical plate exhibit poorly elaborated bipolar shapes, here named 'simple-fusiform' cells. They constitute the vast majority of labeled cells up to P0, and thereafter they decrease in number until P4. As a result of continuous outgrowth of new primary dendrites emerging from the apical pole but also from the basal and lateral aspects of the cell bodies, the Purkinje cells enter the 'complex-fusiform' phase, which peaks by P1 and slowly disappears by P6. The disappearance of 'complex-fusiform' cells is the result of an intense regressive process with resorption or retraction of the long dendrites that reaches a maximum by P3. We have called this stage: the Purkinje cell with 'regressive-atrophic' dendrites. This regression marks the initiation of the phase of the stellate cell, characterized by the explosive outgrowth of shorter perisomatic protrusions emerging in all directions. By P6, almost all the labeled Purkinje cells have attained this phase. The ultrastructural study of the labeled Purkinje cells has revealed that the transient dendrites of the fusiform cells have all the cytologic features of mature dendrites, particularly cytoskeletal elements (microtubules) and free polyribosomes. More importantly, axon terminals of unknown origin establish a few, constantly present, mature-like synaptic contacts on the dendritic shafts and spinous protrusions from P0, the earliest studied age. Their frequency increases on the Purkinje cells which enter the phase of stellate cell. Our results emphasize that the transformation of bipolar postmigratory Purkinje cells into the stellate cell stage results from a complex cascade of alternating creative and destructive processes, taking place in parallel with the formation and regression of mature synaptic contacts, between the remodelling dendritic arbors and unidentified afferent inputs. Purkinje cells, in all the different transitional stages, are present side by side in the same folial regions, at least until P4, and receive a similar contingent of synaptic input. This indicates that the dendritic remodelling is not driven by the synaptic inputs, but obeys either neural interactions that lead Purkinje cells to assume their monocellular layer configuration, or an internal clock depending on the Purkinje cell birthdate, or an interplay between these two kinds of mechanisms.

The nucleotide sequence of the entire Escherichia coli edd-eda region that encodes the enzymes of the Entner-Doudoroff pathway was determined. The edd structural gene begins 236 bases downstream of zwf. The eda structural gene begins 34 bases downstream of edd. The edd reading frame is 1,809 bases long and encodes the 602-amino-acid, 64,446-Da protein 6-phosphogluconate dehydratase. The deduced primary amino acid sequences of the E. coli and Zymomonas mobilis dehydratase enzymes are highly conserved. The eda reading frame is 642 bases long and encodes the 213-amino-acid, 22,283-Da protein 2-keto-3-deoxy-6-phosphogluconate aldolase. This enzyme had been previously purified and sequenced by others on the basis of its related enzyme activity, 2-keto-4-hydroxyglutarate aldolase. The data presented here provide proof that the two enzymes are identical. The primary amino acid sequences of the E. coli, Z. mobilis, and Pseudomonas putida aldolase enzymes are highly conserved. When E. coli is grown on gluconate, the edd and eda genes are cotranscribed. Four putative promoters within the edd-eda region were identified by transcript mapping and computer analysis. P1, located upstream of edd, appears to be the primary gluconate-responsive promoter of the edd-eda operon, responsible for induction of the Entner-Doudoroff pathway, as mediated by the gntR product. High basal expression of eda is explained by constitutive transcription from P2, P3, and/or P4 but not P1.

The role of progesterone (P4) in the regulation of inflammatory mediators interleukin-8 (IL-8), monocyte chemoattractant protein-1, and cyclooxygenase-2 (COX-2) and in the recruitment of leukocyte subpopulations in the endometrium has been examined, by employing a model of P4 withdrawal and maintenance in vivo. Messenger RNA and protein expression have been investigated in endometrial biopsies: 1) during the midsecretory phase (LH+8 to 10); during the maintained luteal phase (P4 administered vaginally for 4 days from LH+8) and biopsies collected 2) 24 h and 3) 48 h post withdrawal of P4; and 4) during pseudo pregnancy (lifespan of corpus luteum extended by 7 days with CG; (decidua collected from women with 5) an ectopic gestation and 6) from women undergoing first-trimester termination of pregnancy). CD56+ large granular lymphocytes remain the major leukocyte subtype, both 24 and 48 h after P4 withdrawal, and in decidua (CG supported or ectopic). Higher numbers (P < 0.05) of macrophages (CD68+) were present in endometrium 48 h post P4 withdrawal and in pseudo pregnant endometrium, compared with normal decidua. Significantly more macrophages (P < 0.01) were present in decidua from an ectopic pregnancy. A significant elevation of IL-8 (P < 0.01) and COX-2 (P < 0.05) messenger RNA was detected 48 h post P4 withdrawal. Evidence is provided for up-regulation of IL-8 and COX-2 in response to P4 withdrawal.

Oligopeptides containing the consensus retroviral protease cleavage sequence Ser/Thr-X-Y-Tyr/Phe-Pro are substrates for purified recombinant HIV-1 protease with Km's in the millimolar range. The minimum sequence containing the consensus pentapeptide which serves as a good substrate is a heptapeptide spanning the P4-P3' residues. Substitution of reduced Phe-Pro or Tyr-Pro dipeptide isosteres or the statine analog 3-hydroxy-4-amino-5-phenylpentanoic acid for the scissile dipeptide afforded inhibitors of HIV-1 protease with Ki values in the micromolar range, three orders of magnitude better in affinity than the corresponding substrates. Inhibitors of HIV-1 protease may provide a novel and potentially useful therapeutic approach to the treatment of acquired immune deficiency syndrome (AIDS).

Tyrosine protein kinases trk, trkB and trkC are essential components of the high affinity receptors necessary to mediate biological effects of the neurotrophins NGF, BDNF, NT-3 and NT-4. Here we report on the expression of these receptors during postnatal development in the rat brain. Cells expressing mRNAs encoding different members of the trk family were identified by in situ hybridization using oligonucleotides complementary to their respective mRNA. In septum, striatum and brainstem, higher levels of trk mRNA were detected at 2 and 4 weeks than at 1 weeks of age. In thalamic nuclei associated with the limbic system, trkB and trkC mRNA were highly expressed at P1 to P7, but the expression declined gradually in 2 and 4 week old animals. Other structures where a developmentally regulated expression was seen included the tenia tecta and piriform cortex where trkB mRNA was not detected until 2 weeks of age. A high labeling was found for trkC mRNA in the deeper parts of neocortex in P1 and P4 animals, while in 2 and 4 weeks old animals the highest labeling was seen over the outer neocortical layers. Several brainstem nuclei showed a higher labeling for trkC mRNA at P1 to P7 than in animals of older age. These data show that expression of members of the trk family is developmentally regulated during postnatal brain development and suggest that high affinity neurotrophin receptors mediate a transient response to neurotrophins in many regions during brain ontogeny.

Four polycyclic aromatic hydrocarbon (PAH)-degrading bacteria, namely Arthrobacter sulphureus RKJ4, Acidovorax delafieldii P4-1, Brevibacterium sp. HL4 and Pseudomonas sp. DLC-P11, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene. [9-14C]Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of 14CO2 was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to 14CO2 by RKJ4, P4-1, HL4 and DLC-P11, respectively. When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in 14CO2 production from [9-14C]phenanthrene in the cases of RKJ4 and HL4, respectively, whereas P4-1 and DLC-P11 did not show any enhancement in 14CO2 production. Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of [9-14C]phenanthrene was observed in mixed culture involving all four microorganisms together. However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in 14CO2 production. In another experiment, the overall growth rate of P4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ4, HL4 and DLC-P11 did not show any enhancement in growth. Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ4 and o-phthalic acid was detected in the case of P4-1. A new intermediate, 1-naphthol, was detected in the cases of HL4 and DLC-P11. HL4 degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P11 degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid. Both transformation sequences are novel and have not been previously reported in the literature. Mega plasmids were found to be present in RKJ4, HL4 and DLC-P11, but their involvement in phenanthrene degradation could not be established.

This study provides a detailed anatomical description of the relation between olivo-cortico-nuclear modules of the intermediate cerebellum of the rat and the intrinsic zebrin pattern of the Purkinje cells. Strips of climbing fibers were labeled using small injections of biotinylated dextran amine into either the medial or dorsal accessory olives, while, in some cases, simultaneous retrograde labeling of Purkinje cells was obtained using gold-lectin injections into selected parts of the interposed nuclei. Our data are represented in a new, highly detailed, cortical surface reconstruction of the zebrin pattern and in relation to the collateral labeling of the climbing fibers to the cerebellar nuclei. We show that the somatotopic regions of the dorsal accessory olive behave differently in their projections to essentially zebrin-negative regions that represent the C1 and C3 zones of the anterior and posterior parts of the cortex. The rostral part of the medial accessory olive projects to zebrin-positive areas, in particular to the P4+ band of the anterior lobe and lobule VI and to the P5+ band of the posterior lobe, indicating that C2 has two noncontiguous representations in the SL and crus 1. By relating the areas of overlap that resulted from the injections in the accessory olives, i.e., labeling of climbing fiber strips and patches of climbing fiber nuclear collaterals, with the results from the injections in the interposed nuclei, i.e., retrograde labeling of Purkinje cells and of inferior olivary neurons, direct verification of the concept of modular cerebellar connections was obtained.

A pathogenicity island present only in highly pathogenic strains of Yersinia (Y. enterocolitica 1B, Y. pseudotuberculosis I and Y. pestis) has been identified on the chromosome of Yersinia spp. and has been designated High-Pathogenicity Island (HPI). The Yersinia HPI carries a cluster of genes involved in the biosynthesis, transport and regulation of the siderophore yersiniabactin. The major function of this island is thus to acquire iron molecules essential for in vivo bacterial growth and dissemination. The presence of an integrase gene and att sites homologous to those of phage P4, together with a G + C content much higher than the chromosomal background, suggests that the HPI is of foreign origin and has been acquired by chromosomal integration of a phage. The HPI can excise from the chromosome of Y. pseudotuberculosis and is found inserted into any of the three copies of the asn tRNA loci present in this species. A unique characteristic of the HPI is its wide distribution in various enterobacteria. Although first identified in Yersinia spp., it has subsequently been detected in other genera such as E. coli, Klebsiella and Citrobacter.

Uterine glands and their secretions are required for conceptus (embryo/fetus and associated placenta) survival and development. In most mammals, uterine gland morphogenesis or adenogenesis is a uniquely postnatal event; however, little is known about the mechanisms governing the developmental event. In sheep, progestin treatment of neonatal ewes permanently ablated differentiation of the endometrial glands. Similarly, progesterone (P4) inhibits adenogenesis in neonatal mouse uterus. Thus, P4 can be used as a tool to discover mechanisms regulating endometrial adenogenesis. Female pups were treated with sesame vehicle alone as a control or P4 from Postnatal Day 2 (PD 2) to PD 10, and reproductive tracts were examined on PD 5, 10, or 20. Endometrial glands were fully developed in control mice by PD 20 but not in P4-treated mice. All other uterine cell types appeared normal. Treatment with P4 stimulated proliferation of the stroma but suppressed proliferation of the luminal epithelium. Microarray analysis revealed that expression of genes were reduced (Car2, Fgf7, Fgfr2, Foxa2, Fzd10, Met, Mmp7, Msx1, Msx2, Wnt4, Wnt7a, Wnt16) and increased (Hgf, Ihh, Wnt11) by P4 in the neonatal uterus. These results support the idea that P4 inhibits endometrial adenogenesis in the developing neonatal uterus by altering expression of morphoregulatory genes and consequently disrupting normal patterns of cell proliferation and development.

Activation of glutamatergic synapses results in long-lasting sodium transients in astrocytes mediated mainly by sodium-dependent glutamate uptake. Sodium elevations activate Na(+) /K(+) -ATPase and glucose uptake by astrocytes, representing key signals for coupling glial metabolism to neuronal activity. Here, we analyzed the spread of sodium signals between astrocytes in hippocampal slice preparations. Stimulation of a single astrocyte resulted in an immediate sodium elevation that spread to neighboring astrocytes within a distance of ∼ 100 μm. Amplitude, slope, and propagation speed of sodium elevations in downstream cells decayed monotonically with increasing distance, indicative of a diffusion process. In contrast to sodium, calcium increases elicited by electrical stimulation were restricted to the stimulated cell and a few neighboring astrocytes. Pharmacological inhibition of mGluR1/5 slightly dampened the spread of sodium, whereas inhibition of glutamate uptake or purinergic receptors had no effect. Spread of sodium to neighboring cells was disturbed on pharmacological inhibition of gap junctions, reduced in animals at P4 and virtually omitted in Cx30/Cx43 double-deficient mice. In contrast to results obtained earlier in cultured astrocytes, our data thus indicate that calcium signaling and metabotropic glutamate receptors are supportive of, but not prerequisites for, the spread of sodium between hippocampal astrocytes in situ, whereas expression of Cx30 and Cx43 is essential. Cx30/Cx43-mediated sodium diffusion between astrocytes could represent a signal indicating increased metabolic needs, independent of concomitant calcium signaling. Spread of sodium might also serve a homeostatic function by supporting the re-establishment of steep sodium gradients and by lowering the metabolic burden imposed on single cells.