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Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality "foundational datasets" consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010-2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private users to upload and analyze sensitive data, and for collating cross-platform datasets to identify and validate congruent datapoints. SNP datasets from WGS assemblies and pan-genome microarrays are analyzed in a combinatorial fashion to determine relatedness of 33 Salmonella enterica strains to six strains collected as part of an outbreak investigation. Use-case scenario three utilizes published surveillance results that describe the incidence and sources of O157:H7 E. coli isolates associated with a produce pre-harvest surveillance study that occurred during 2002-2006. In summary, PATRN is a web-based integrated platform containing tools for the management, analysis and visualization of data about foodborne pathogens., (Published by Elsevier Ltd.)

Background: Members of the genus Cronobacter are causes of rare but severe illness in neonates and preterm infants following the ingestion of contaminated infant formula. Seven species have been described and two of the species genomes were subsequently published. In this study, we performed comparative genomics on eight strains of Cronobacter, including six that we sequenced (representing six of the seven species) and two previously published, closed genomes., Results: We identified and characterized the features associated with the core and pan genome of the genus Cronobacter in an attempt to understand the evolution of these bacteria and the genetic content of each species. We identified 84 genomic regions that are present in two or more Cronobacter genomes, along with 45 unique genomic regions. Many potentially horizontally transferred genes, such as lysogenic prophages, were also identified. Most notable among these were several type six secretion system gene clusters, transposons that carried tellurium, copper and/or silver resistance genes, and a novel integrative conjugative element., Conclusions: Cronobacter have diverged into two clusters, one consisting of C. dublinensis and C. muytjensii (Cdub-Cmuy) and the other comprised of C. sakazakii, C. malonaticus, C. universalis, and C. turicensis, (Csak-Cmal-Cuni-Ctur) from the most recent common ancestral species. While several genetic determinants for plant-association and human virulence could be found in the core genome of Cronobacter, the four Cdub-Cmuy clade genomes contained several accessory genomic regions important for survival in a plant-associated environmental niche, while the Csak-Cmal-Cuni-Ctur clade genomes harbored numerous virulence-related genetic traits.

Cronobacter spp. (formerly Enterobacter sakazakii) is an emerging foodborne pathogen consisting of seven species including C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis (with three subspecies, dublinensis, lausannensis, and lactaridi), C. universalis, and C. condimenti. To date, 12 Cronobacter serogroups have been identified. In this study, MboII restriction fragment length polymorphism patterns and DNA sequences of O-antigen gene clusters were used to identify novel serogroups of Cronobacter spp. Sequence analysis of the O-antigen regions, located between galF and gnd, of strains with distinct restriction fragment length polymorphism patterns revealed five unique gene clusters. These new O-antigen gene clusters were species specific and were termed C. turicensis O3, C. muytjensii O2, C. dublinensis O1, C. dublinensis O2, and C. universalis O1. Polymerase chain reaction assays were developed using primers specific to O-antigen processing genes and used to screen a collection of Cronobacter strains to determine the frequency of these newly identified serotypes.

In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations.

Cronobacter spp. are emerging pathogens that cause severe infantile meningitis, septicemia, or necrotizing enterocolitis. Contaminated powdered infant formula has been implicated as the source of Cronobacter spp. in most cases, but questions still remain regarding the natural habitat and virulence potential for each strain. The iron acquisition systems in 231 Cronobacter strains isolated from different sources were identified and characterized. All Cronobacter spp. have both the Feo and Efe systems for acquisition of ferrous iron, and all plasmid-harboring strains (98%) have the aerobactin-like siderophore, cronobactin, for transport of ferric iron. All Cronobacter spp. have the genes encoding an enterobactin-like siderophore, although it was not functional under the conditions tested. Furthermore, all Cronobacter spp. have genes encoding five receptors for heterologous siderophores. A ferric dicitrate transport system (fec system) is encoded specifically by a subset of Cronobacter sakazakii and C. malonaticus strains, of which a high percentage were isolated from clinical samples. Phylogenetic analysis confirmed that the fec system is most closely related to orthologous genes present in human-pathogenic bacterial strains. Moreover, all strains of C. dublinensis and C. muytjensii encode two receptors, FcuA and Fct, for heterologous siderophores produced by plant pathogens. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed which genes and operons are components of the Fur regulon. Taken together, these results support the proposition that C. sakazakii and C. malonaticus may be more associated with the human host and C. dublinensis and C. muytjensii with plants.

Growth of two Vibrio tubiashii strains under iron-chelated conditions resulted in the production of a hydroxymate-like siderophore, and expression of outer membrane proteins with homologies to proteins in Vibrio cholerae and Vibrio vulnificus which were not seen in cells grown under non-chelated growth conditions. PCR analysis using primers based on Listonella anguillarum's ferric uptake Repressor protein (fur) gene detected a 316 bp fur gene homolog which also had sequence homology to the fur genes of V. cholerae and V. vulnificus. V. tubiashii cultured under iron-chelated growth conditions induced a greater fluid accumulation (FA) response in suckling mice than cells which were cultured under iron non-chelated growth conditions. Our observations that V. tubiashii possesses a fur-like gene homolog and expresses unique OMPs, a hydroxymate-like siderophore, and produces an increased fluid accumulation response in suckling mice when grown under iron-chelated condition support previous findings that V. tubiashii may have the essential components for the survival and establishment of infections and this report represents the first observations of competent iron acquisition system in V. tubiashii which is similar to those produced by other marine vibrios, many of which are pathogenic for humans., (Published by Elsevier Ltd.)

Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes.

Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

Reactome (http://www.reactome.org) is a collaboration among groups at the Ontario Institute for Cancer Research, Cold Spring Harbor Laboratory, New York University School of Medicine and The European Bioinformatics Institute, to develop an open source curated bioinformatics database of human pathways and reactions. Recently, we developed a new web site with improved tools for pathway browsing and data analysis. The Pathway Browser is an Systems Biology Graphical Notation (SBGN)-based visualization system that supports zooming, scrolling and event highlighting. It exploits PSIQUIC web services to overlay our curated pathways with molecular interaction data from the Reactome Functional Interaction Network and external interaction databases such as IntAct, BioGRID, ChEMBL, iRefIndex, MINT and STRING. Our Pathway and Expression Analysis tools enable ID mapping, pathway assignment and overrepresentation analysis of user-supplied data sets. To support pathway annotation and analysis in other species, we continue to make orthology-based inferences of pathways in non-human species, applying Ensembl Compara to identify orthologs of curated human proteins in each of 20 other species. The resulting inferred pathway sets can be browsed and analyzed with our Species Comparison tool. Collaborations are also underway to create manually curated data sets on the Reactome framework for chicken, Drosophila and rice.

Biological Pathway Exchange (BioPAX) is a standard language to represent biological pathways at the molecular and cellular level and to facilitate the exchange of pathway data. The rapid growth of the volume of pathway data has spurred the development of databases and computational tools to aid interpretation; however, use of these data is hampered by the current fragmentation of pathway information across many databases with incompatible formats. BioPAX, which was created through a community process, solves this problem by making pathway data substantially easier to collect, index, interpret and share. BioPAX can represent metabolic and signaling pathways, molecular and genetic interactions and gene regulation networks. Using BioPAX, millions of interactions, organized into thousands of pathways, from many organisms are available from a growing number of databases. This large amount of pathway data in a computable form will support visualization, analysis and biological discovery.

Sagar Island, situated in the east coast of India and one of the biggest deltas in Sundarban group, faces coastal erosion and degradation of coastal vegetation and various natural hazards. Erosion is mainly due to clay mining, wave activities, and the impact of river and tidal currents of Muri Ganga and Hugly Rivers. Further, the coastal zone of Sagar Island faces increasingly severe problems of rapidly growing human population, deteriorating environmental quality, and loss of critical habitats. Sagar Island has been victimized several times by tropical cyclones and influenced daily by tidal fluctuations. The island needs immediate attention on the coastal zone in order to protect the shoreline and ecosystem. The capability of satellite remote sensing to provide synoptic, repetitive, and multispectral data has proved to be very useful in the inventory and monitoring of critical coastal issues. Sagar Island and its environs are subjected to both natural and anthropogenic activities that continuously modify the region.

Reactome (http://www.reactome.org) is an expert-authored, peer-reviewed knowledgebase of human reactions and pathways that functions as a data mining resource and electronic textbook. Its current release includes 2975 human proteins, 2907 reactions and 4455 literature citations. A new entity-level pathway viewer and improved search and data mining tools facilitate searching and visualizing pathway data and the analysis of user-supplied high-throughput data sets. Reactome has increased its utility to the model organism communities with improved orthology prediction methods allowing pathway inference for 22 species and through collaborations to create manually curated Reactome pathway datasets for species including Arabidopsis, Oryza sativa (rice), Drosophila and Gallus gallus (chicken). Reactome's data content and software can all be freely used and redistributed under open source terms.

Reactome http://www.reactome.org, an online curated resource for human pathway data, provides infrastructure for computation across the biologic reaction network. We use Reactome to infer equivalent reactions in multiple nonhuman species, and present data on the reliability of these inferred reactions for the distantly related eukaryote Saccharomyces cerevisiae. Finally, we describe the use of Reactome both as a learning resource and as a computational tool to aid in the interpretation of microarrays and similar large-scale datasets.

Spatial distribution of pH, electrical conductivity (EC), total dissolved solids (TDS), fluoride and total iron content of ground water samples collected from the muvattupuzha river basin, Kerala, India, has been studied for pre monsoon and post monsoon periods of year 2001. Results showed the groundwater of the basin is acidic for which the pH values ranged between 5.5 and 8.0. Average EC was found to be less than 100 microS/cm, for most of the study region. The pre monsoon minimum and maximum TDS were found as 25.6 and 227.84 mg/L respectively, where as post monsoon values ranged between 16 and 162.56 mg/L. The relatively low EC and TDS values found both during the seasons in the lateritic terrain of the river basin signifies the lower residence time of ground water with the country rock. This makes the groundwater quality of this river basin as good. Pre monsoon season samples showed high total iron content than that during the post monsoon period. During the study period values of the fluoride contents were found to be within the permissible limits.

Background: Free radical mediated injury is increasingly recognized in many metabolic diseases including Wilson's disease (WD). Use of antioxidants as an adjunctive therapy in WD may have therapeutic significance., Aim: The aim of the study was to correlate serum levels of tocopherols with serum copper and ceruloplasmin and clinical status of these patients., Methods: Serum levels of tocopherol of were measured spectrophotometrically using the Emmerie-Engel reaction in 34 patients from a large cohort of WD being followed up at a tertiary care center., Results: Majority of patients were male (M/F=23:11). The mean serum copper was 43.6+/-26.2 microg/dl (range=10-121 microg/dl) and serum ceruloplasmin was 5.6+/-5.5 mg/dl (range=0-30 mg/dl). The mean serum tocopherol level was 0.68+/-0.18 mg/dl (range=0.23-1.14 mg/dl) and compared to the control (1.07+/-0.17 mg/dl), nearly 59% of patients had decreased levels (p<0.001). No significant correlation was noted between low serum tocopherol levels and serum copper levels, Mini Mental Status Examination (MMSE) scores and CHU staging. However, serum tocopherol levels were lower in patients with relatively short duration of treatment (7.8 years vs. 12.4 years)., Conclusion: Decreased levels of serum tocopherol were detected in 59% of patients compared to controls. However, low tocopherol levels did not correlate with clinical status or biochemical parameters of WD, except for relatively shorter duration of treatment. Further studies, especially in newly diagnosed patients, need to be done to validate the role of low tocopherol levels in Wilson's disease.

There is an emerging evidence that plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) may have potential as a chemotherapeutic agent. However, the growth inhibitory mechanisms of plumbagin have remained unexplored. The aim of the study was to determine whether plumbagin-induced cell death in human cervical cancer cell line, ME-180, exhibited biochemical characteristics of apoptosis and to check whether N-acetyl-l-cysteine (NAC), which is a free radical scavenger, can reverse the cytotoxic effects of plumbagin. It can be concluded from the results that plumbagin inhibits the growth of ME-180 cells in a concentration and time-dependent manner. The cytotoxic effect of plumbagin induced cell death is through the generation of reactive oxygen species (ROS) and subsequent induction of apoptosis as demonstrated by the present data. Treatment of cells with plumbagin caused loss of mitochondrial membrane potential (DeltaPsi(m)), and morphological changes characteristic of apoptosis, such as the translocation of phosphatidyl serine, nuclear condensation, and DNA fragmentation. Moreover, plumbagin-induced apoptosis involved release of mitochondrial cytochrome c and apoptosis inducing factor (AIF), thus activation of caspase-dependent and -independent pathways, as shown by the plumbagin-mediated activation of caspase-3 and -9. Our results also show that pretreatment of ME-180 cells with NAC blocks plumbagin-induced loss of DeltaPsi(m) and subsequent release of cytochrome c, AIF, and caspase-9 and -3 activation, thus inhibiting the apoptotic ability of plumbagin.

The quantification of the tumour volume is essential for the assessment of therapy-induced changes. Traditional methods of assessing the response of neuroendocrine tumours using radiological methods yield poor results, particularly within the liver. The aim of this study was to establish whether it would be possible to identify a method using functional volumes to predict the response of tumours to various therapies. Twenty-two patients with neuroendocrine tumours of carcinoid type in the liver were treated with chemotherapy, chemo-embolization or 90Y-radiolabelled somatostatin analogues. All patients underwent 111In-pentetreotide single-photon emission computed tomography (SPECT) and computed tomography (CT) scan pre- and post-treatment. The tumour functional volume, a measure of metabolically active tumour tissue, was calculated from the SPECT images using a 10-point display; regions of interest were drawn around 50% of the maximum tumour activity, slice by slice, and then multiplied by the slice thickness (9.3 mm). Any difference in functional volume was compared with the CT response, using the Response Evaluation Criteria in Solid Tumours (RECIST), and clinical outcome. At 6 months after treatment, 14 patients showed a good clinical response, as measured by a reduction in pain, flushing or abdominal symptoms; the functional volume of the tumours in these patients decreased by a mean of 25% (range, 1-52%). Of the eight patients who showed no symptomatic relief, or in whom symptoms worsened, the functional volume increased by a mean of 74%. Using a change in functional volume of more than 25% as significant, SPECT predicted 13 of the 22 (59%) clinical outcomes correctly; if a 10% change was used, 18 of the 22 (81.1%) clinical outcomes were correctly predicted. However, CT, using RECIST, only predicted eight of the 22 (36%) clinical outcomes correctly. The assessment of the total functional volume by SPECT quantification is more useful than CT in monitoring tumour response after treatment, and the changes in functional volumes after therapy correlate well with the clinical response.

Previous studies have shown that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally act as a growth inhibitor in these cells. Also, there are other reports that suggest that wild-type BRCA1 protein may repress estrogen receptor (ER) function either directly or indirectly. However, response to antiestrogen drugs in BRCA1-blocked ER-positive ovarian cancer cells has not been reported, and this served as the rationale for this study. We analyzed the effect of tamoxifen, emodin, and plumbagin in BRCA1-blocked ER-positive BG-1 ovarian cancer cells. For all three drugs, BRCA1-blocked cells were more sensitive than the corresponding control cells as assessed by MTT assay; however, only plumbagin showed a statistically significant difference in mean viability (P < 0.05). All three drugs induced loss of mitochondrial membrane potential (DeltaPsi(m)), nuclear condensation, DNA fragmentation, and morphological changes, as observed after 6 h of drug treatment, suggesting apoptosis induction in both BRCA1-blocked and control cells. However, apoptosis induction was greater in BRCA1-blocked cells, the efficacy being in the order of plumbagin > tamoxifen > emodin. The dose of plumbagin needed to kill 50% was 5 microM in the control cells and 2.68 microM for the BRCA1-blocked cells, indicating that the latter was about twofold more sensitive to plumbagin than the wild-type cells. This throws light on the fact that plumbagin may have chemotherapeutic potential as an anticancer agent in BRCA1-mutated ovarian cancer patients., (Copyright 2003 Wiley-Liss, Inc.)

Vertebro basilar insufficiency (VBI) is a well known cause of vertigo. Brain Single Photon Emission Computed Tomography (SPECT) is an important diagnostic tool to detect and to quantitate the perfusion abnormalities in different areas of the brain. Effect of an antivertigo drug Betahistine on improving the hypoperfusion in different areas of the brain in vertigo patients was studied using brain SPECT. Betahistine at a dose of 16 mg three times daily was shown to improve perfusion in the hypoperfused areas of the brain resulting in relief from symptoms of vertigo. The cerebellar region, which is the most important area involved in vertigo patients with vascular pathology, showed almost complete normalisation of perfusion following Betahistine therapy.

In this essay, I demonstrate through numerous examples taken from four identifiable Hindi film subgenres queer themes which, though nontransgressive in their native Indian context, acquire subversive value and serve as queer points of identification when viewed from a non-nationalist bias. Watching particular films with this "queer diasporic viewing practive," sex/gender play which is normative (yet still coded) in the land of the films' production can be reclaimed as queer through the differently subjective lens of transnational spectatorship, a lens removed from patriarchy, sexism, and homophobia. This particularly becomes apparent in the Bollywood dance sequence-the frequent site of Hindi sex/gender play-whose coded queer desires are much easier to de-code (or re-code) when in the diaspora.

Changes in sleep after fetal preoptic (POA) tissue transplantation were studied in rats which had been made insomniac by a medial preoptic area (mPOA) lesion. Two days after the N-methyl D-aspartic acid (NMDA) lesion of the mPOA, fetal POA tissues (obtained from 14- to 17-day-old fetuses) were transplanted into the lesioned mPOA. Insomnia was less marked in these animals, as compared to nontransplanted lesioned rats, even on the 4th day after transplantation. The quantum of sleep nearly attained the prelesion level by the 20th day. Body weight also showed recovery after transplantation. Rectal temperature, which was increased by the lesion of the mPOA, remained unaltered even after the transplantation. These results suggest that the recovery of sleep and rectal temperature may follow different time courses. Surviving transplanted neurons were seen at the site of lesion on postmortem examination. Humoral interaction between the host and the transplant may be responsible for the early recovery of sleep, though the establishment of neural connections between the host and transplant might have contributed to the later recovery. This is the first study to show the recovery of sleep function in insomniac animals after fetal preoptic tissue transplantation. However, the specificity of the POA fetal tissue, in comparison with other neural tissues to promote sleep recovery, remains to be established.

The role of the medial preoptic area (mPOA) beta adrenergic receptors in the regulation of sleep-wakefulness (S-W) was investigated in this study. S-W was assessed on the basis of polygraphic recording of EEG, EMG and EOG in free moving rats. Intracerebral microinjection of beta agonist, isoproterenol, into the mPOA produced arousal. The study was also conducted on another set of rats in which noradrenergic (NE) innervation to the mPOA was destroyed by injecting 6-hydroxydopamine into the ventral noradrenergic bundle, in the brain stem. Local application of isoproterenol, into the mPOA, in these animals, did not produce any significant change in S-W. Thus, the increase in awake period obtained on isoproterenol administration was the result of its action on the presynaptic NE terminals. Possible involvement of other responses in the isoproterenol induced increase in wakefulness, is discussed.

Fetal ventral mesencephalon from the 15th gestational day was grafted into the striatum of neonatal and adult rats. In one group of adult rats, fetal nigra was transplanted into normal striatum. In a second group, the tissue was transplanted at sites where dopaminergic fibers were denervated with 6-hydroxydopamine. The behavior of the dopaminergic neurons and glial reactions were studied by staining with cresyl violet to localize the transplants and by immunolabeling tyrosine hydroxylase (TH) and glial fibrillary acidic protein. In normal adults, the transplants were small. At the edge of the transplants, TH-positive neurons were packed into clusters, and an interface without any significant crossover of TH-positive fibers was present. Glial reaction was minimal in and around the transplant. In the denervated striatum, transplants were generally larger than those in normal striatum and surrounded by a glial scar. TH-positive neurons were both closely packed and loosely arranged at the periphery of the transplants. Processes could be clearly defined and could be traced to the adjacent host striatum through the TH-free denervated area. In neonates, the transplants were large and at times extended beyond the striatum. Most TH-positive neurons were arranged linearly along the periphery of the transplant. Cell bodies were widely separated and a well-developed neuropil was present. Fibers from the transplant mingled freely with the host striatum without any interface. In all three transplant groups, tracing the TH-positive neurites was easy because they were thicker and coarser than other elements. No apparent glial reaction occurred in the neonates. Thus, the growth and maturation of dopaminergic neurons seemed to vary in different environments. The most conducive environment appears to be neonatal brain in which growth factors are readily available.

Electron microscopy of the maturing neurons and developing and maturing synapses in the substantia nigra of 14 human embryos/foetuses of 8-24 weeks of gestation are reported. At 8 weeks, cells were immature with very little cytoplasm and cellular organelles. Contact sites of processes appeared more electron dense than the other areas. At 12 weeks, many of the cells had acquired more cytoplasm and cellular organelles and could be identified as neurons. Asymmetric synapses with clear, round synaptic vesicles also were identifiable at this age. Such synapses, first to appear in the developing substantia nigra, are reported to be formed by recurrent collateral nigro-striatal fibres. Substance P fibres from the striatum also are contributing to this type of synapse. At 15-16 weeks, not only was the number of such synapses increased, but many appeared morphologically mature. Symmetric synapses having clear round vesicles along with a few dense core vesicles also appeared at this stage, suggesting striatal input. By 24 weeks of gestation, most of the neurons had cytological features comparable to that of the mature neurons. There was an increase in the total number of synapses and the individual variety from 15 to 24 weeks of gestation. The present study indicates that synaptogenesis starts at 8 weeks and continues beyond 24 weeks of gestation.

The substantia nigra of gestation day 14 was transplanted into the striatum of 3-4-month-old rats to investigate the transplants ultrastructurally at the end of 2 years, as a follow-up to our previous studies. Transplants were of small size in all 10 specimens taken for this study. The changes observed in the transplant and in the interface region with the host striatum were: thickening of the blood vessel walls, perivascular cuffing with lymphocytes and macrophages loaded with tissue debris, degenerating neurons and hypertrophied astroglia containing dense granules indicating ageing or reaction to degeneration and glial processes. The number of surviving neurons in the transplants was small. These were smaller in size and had very few intracytoplasmic membraneous organelles. A higher content of intracytoplasmic ageing lipofuscin pigment was present than in host neurons and age-matched nigral neurons. Synapses were few, and their number varied among transplants. Generally, the synapses were at the interface with the host tissue. The changes observed in all the 2-year-old transplants suggest premature ageing or a slow rejection process. Slow rejection is a possibility, because these rats are only stock-bred, not inbred, and hence they are not completely immunologically compatible.

Background: The crown-rump length is conventionally used to determine the age of human abortuses. However, it is not reliable as it is dependent on the positioning of the conceptus. We compared this with the biparietal diameter and foot length for determining the gestational age., Methods: Different measurements, commonly used to assess gestational age, were measured in 146 human abortuses for which an accurate obstetric history could not be elicited. Measurements taken were crown-rump length, biparietal diameter and foot length. These were correlated with the observations at antenatal examinations before finalizing the approximate age., Results: Multiple regression analysis of the data indicated that of the three measurements, the biparietal diameter was the most reliable for determining foetal gestational age between 8 and 26 weeks. The age determined with the biparietal diameter correlated well with that of abortuses with an accurate obstetric history., Conclusion: The biparietal diameter of a human foetus may be used to determine its age if the obstetric history regarding the period of gestation is vague or not available.

Cell surface molecules, NCAM and L1, reported to have a role in synaptogenesis, growth and fasciculation of the neurites in the brain, were traced in the embryonic nigral transplants in the host striatum of adult rats. Substantia nigra of five, 15 and 25 postnatal days were also examined for the same molecules. Tyrosine hydroxylase label was used as a marker to localize the nigral neurons and glial fibrillary acidic protein to detect if glial scar present. In the control as well as transplants large neurons had expressed tyrosine hydroxylase. By 15th postnatal day tyrosine hydroxylase neurons appeared mature and were scattered, suggesting a well-formed neuropil. NCAM and L1 reaction was seen as a peripheral rim in most of the cells on the fifth postnatal day. The reaction was mainly in relation to the large cells and more extensive on the 15th day. Thereafter on the 25th day, activity was negligible. Large neurons demonstrated strong reactivity for NCAM and L1 during early post-transplantation days. After 30 days only smaller cells were reactive, many of which could be identified as neurons. Strong reaction for these molecules was present only until 60 days, though faint reaction could be detected even on the 90th day. These observations indicate that the growth promoting molecules, the type seen in the neonatal period, can be detected normally only until the neurons mature. Prolonged expression of these molecules by the grafted neurons indicate delay in the maturation of these cells due to absence of adequate target sites for synaptic connections. Some of the smaller cells expressing these molecules after 30 days of transplantation could be astroglia, either proliferating or reactive.

The role of the preoptic area (POA) neurons in the regulation of sleep-wakefulness (S-W) has been investigated in this study. The cell-specific neurotoxin, kainic acid (KA), was injected (0.8 microgram in 0.2 microliter) intracerebrally for lesioning of the POA. S-W was assessed (on the basis of EEG, EMG, and EOG recordings) for a day before bilateral lesion of the POA, and for 3 weeks after the lesion. There was an increase in wakefulness, and a decrease in all the stages of sleep after KA lesion of the POA. The reduction in deep slow wave sleep (S2) and REM sleep (PS) were more marked than light slow wave sleep (S1), and these had not shown any recovery even after 3 weeks of lesion. Two days after the lesion, the reduction in sleep was much more marked during the daytime than at night. There was an increase in locomotor activity, especially during the daytime, though it was only statistically significant on the 6th and the 10th day after the lesion. This study shows that the POA neurons are involved in the induction and maintenance of sleep. The lesion did not have a long lasting effect on the circadian distribution of sleep but the changes in locomotor activity seem to persist for a longer period.

This study confirmed that bilateral kainic acid (KA) injection at the caudate produces aphagia and adipsia in rats. The reduction in food and water intake was fatal after a higher dosage of the drug. To test the effect of transplantation on the mortality rate, KA was first injected in the left caudate, in one set of rats. After a gap of three days, fetal striatal tissue was unilaterally transplanted at this lesioned site, along with a second injection of KA in the right caudate. Successful transplantation, as ascertained morphologically, did not significantly alter the mortality rate. The morphometric study revealed that the neurons of the transplant were larger in size, and their numerical density lower than those of the caudate of normal rats. Only very few neurons of the transplant developed functional connectivity with the host, as demonstrated by electrophysiological studies.

Histological and quantitative studies were carried out in dentate nucleus (DN) of 14 human fetuses of gestational ages ranging from 10 weeks to 28 weeks in Nissl stained sections. The dentate nucleus was already well delineated at 11 weeks, but the adult profile was attained only by 27-28 weeks. The magnocellular dorsomedial region and the parvocellular ventrolateral region became apparent at 17-18 weeks. The numerical density of neurons showed a steady decline with advancing gestational age. However, the absolute neuronal count until 19-20 weeks was 367.1 x 10(3) followed by a count of 249.5 x 10(3) at 22-23 weeks. The reduction in the count between the two periods was significant and coincided with cell death which was striking between 19 and 23 weeks. The glial cell population continued to increase with advancing gestational age. The occurrence of significant neuronal cell death and its probable role in the developing human DN is reported for the first time.

The substantia nigra from rats of 16 and 17 gestational days grafted into the lateral ventricle of adult rats grew into isolated or partially integrated transplants in 80% of the rats. The morphological details of the transplants were studied at intervals of 30-360 days. The neurons grew, differentiated and were comparable to age-matched controls until 150 days. Thereafter, the neurons showed progressive aging changes like dendritic thickening, clear spaces, membrane-bound vacuoles and increase in lysosomes in the cytoplasm. The numerical density per unit volume (Nv) of normal neurons significantly decreased with simultaneous increase in Nv of glial cells by 360 days. These changes were slower to develop in the partially integrated transplants: it is concluded that target tissue interaction is necessary for prolonged survival of the grafted tissue.

Fetal nigral neurons from 16 and 17 gestational days were transplanted into the intact striatum of adult rat. On different post-transplantation days (30-360 days), the structural and immunohistochemical details of the transplants were studied. The grafted neurons matured and showed phenotypical characteristics comparable to that of normal nigral neurons in adult rats until 180 days. Tyrosine hydroxylase-positive neurons were seen not only in the transplant but also in the adjacent host striatum. Tyrosine hydroxylase-positive fibres were also seen extending for a short distance into the host striatum. A large number of synapses in the transplants were of asymmetric type, containing clear round vesicles. These synapses resembled the few intrinsic type present in the normal substantia nigra. On the other hand, the predominant type 2 synapses with pleomorphic vesicles in the normal nigra were infrequently encountered in the transplants. On the 300th day, the cytoplasm of a few of the neurons showed ageing changes in the form of clear spaces, paucity of organelles especially rough endoplasmic reticulum, membrane-bound vacuoles and increase in the lipofuscin population. In addition, localized thickening of the soma and the dendrites were seen in relation to randomly distributed neurons. By 360 days, more than one quarter (26%) of the total neurons showed these changes indicating ageing. The number per unit volume of normal neurons decreased significantly when compared to the transplants on 60 and 90 days. In the substantia nigra of age-matched control, except for an increase in the lysosomal population, other ageing changes were not detectable. The neurons of intact substantia nigra of the host rat, chronologically 4-8 months older than the transplanted neurons, also appeared normal but for lipofuscin granules. The present study provides morphological evidence for rapid ageing of neurons in the long term nigral transplants. These observations raise fresh doubts regarding permanent survival of grafted neurons in the host brain. Studies so far conducted are after prior nigral lesions. Trophic factors following lesions of the host tissue may have influenced the long term survival of the transplanted neurons. On the other hand, such changes may have been missed since no detailed morphological investigations of the long term transplants have been done so far.

The neurobiological basis for the short-term recovery in Parkinson's patients and experimental animals grafted with adrenal medulla is not yet clear. Structural details of the grafted chromaffin cells are also not available. In the present study, autografts of adrenal medulla in the anterior eye chamber, lateral ventricle and striatum of adult rats were studied for 360 days. Though a large number of cells degenerated, a few healthy chromaffin cells survived up to 360 days in the anterior eye chamber. In the ventricular and striatal regions, cells degenerated more rapidly, and a few surviving cells were seen only up to 120 and 150 days, respectively. Degeneration of the cells was evident from the alteration of the cytoplasmic granules, appearance of vacuoles and lysosomes, rapid decline in the number of TH and DBH positive cells and diffusion of enzymes in the intercellular region. Only lymphocytes and connective tissue cells were seen in the ventricle after 120 days, while outlines of a few chromaffin cells and background fluorescence were still evident in the striatum up to 150 days. In some of the intrastriatal transplants, morphologically identifiable Schwann cells were present and, in one transplant, there was evidence of myelination of axons by Schwann cells. These axons were obviously derived from the adjacent host tissue. From the findings it has been concluded that autografts of adrenal medulla survive for only a limited period of time on transplantation to the central nervous system and anterior eye chamber. Survival seemed to be better in the anterior eye chamber than at the sites preferentially chosen for treating Parkinson's patients or experimental animals.

Substantia nigral grafts of 16 and 17 gestation days showed phenotypic characteristics in the anterior eye chamber of adult rats until the third month after transplantation. Thereafter by the sixth month a number of neurons showed somal and dendritic thickening, reduced population of endoplasmic reticulum, increase in lysosomes, and clear areas devoid of organelles, indicating age changes. These changes were progressive and affected more neurons by the end of 1 year, the longest period studied. The observations suggest that the maturation of nigral neurons is independent of specific afferent input, whereas target influence is necessary for the continued maintenance of the mature neurons. All the synapses observed in the transplant were of the asymmetric variety, reminiscent of the few intrinsic synapses of the intact nigra. This suggested establishment of mutual connectivity among the transplanted neurons in the absence of a target and the type of synapse formed may have been influenced by the local environment. Large glial processes, very prominant during the 4- to 6-month period became less significant afterward but continued to be present until the end of the period studied. Though there was no morphological evidence of lymphocytic infiltration, this might suggest an immunologic reaction.

Undernutrition of the newborn rats, produced during the first 3 weeks by increasing the litter size and restricting the mother's diet, resulted in reduction of the body and brain weights of the experimental animals. One group of undernourished animals showed especially severe reduction of body and cerebellar weights. These animals, on the 10th postnatal day, had an immature cerebellar cortex corresponding to that of the 7th day postnatal control animals. The external granular layer persisted in the cerebellar cortex of the underweight animals until the 23rd day, while it disappeared by 20th day in the control animals. Mitotic activity was evident until the 21st postnatal day in these animals while it stopped in the normal animal by 16th postnatal day. There was no marked difference in the fine structure of the various cell types in the control and undernourished animals. Midsagittal tracings of the cerebellar cortex showed a reduced surface area in the undernourished animals, while the thickness of the external granular layer and molecular layer did not show any significant difference when compared to that of the control animals, thus showing a reduction in total cell number, but not per unit area. The normal morphological appearance of the cerebellar cortex in the underfed animals of higher weight probably indicates that these animals are adequately nourished in spite of the reduction in weight when compared to the control animals, which probably are overfed.

Continuous irradiation in utero is reported to produce mental retardation and gross abnormalities of the brain in the human. A few experimental studies conducted so far also report gross brain defects in animals exposed to continuous irradiation in utero. Despite the increasing use of nuclear energy for power and radioisotopes in medicine, there is hardly any literature available on the effect of continuous irradiation on the structural details of the developing brain. After intraperitoneal injections of different doses of 131I (8, 18 and 32 microCi) and 32P (10 microCi) in new-born rats on the 6th postnatal day, cerebella stained by Golgi techniques were cut sagittally and the sections were examined on the 10th, 15th and 21st postnatal days. In the animals injected with 18 and 32 microCi of 131I and 10 microCi of 32P a large number of Purkinje cells showed morphological alterations not seen in the control groups or in the groups injected with 8 microCi of 131I. The changes observed included persistence of the perisomatic processes beyond the 10th postnatal day, multiple primary dendrites, angulation of the primary dendrites, long segments of primary dendrites without branches and significantly reduced dendritic volume. The number of affected cells was less on the 21st postnatal day. The effective radiation dose estimated in these groups ranged from 15 to 26 rad. Since the rats irradiated with 6 rad had not shown such changes it is believed that there is a threshold dose of radiation beyond which only changes are perceptible at neuronal level.

Mice given daily i.p. injections of immunoglobulins against ovine LH on Days 3-7 of pregnancy were devoid of implantation sites on Day 8 whereas mice treated with antibodies to hCG had embryos of normal number and appearance on Day 8. These antibody treatments reduced the mean +/- s.d. serum progesterone concentrations from 65.4 +/- 15.3 ng/ml (control globulins) to 8.6 +/- 4.9 ng/ml (anti-LH) and 9.2 +/- 3.1 ng/ml (anti-hCG) on Day 8 and had no differential effect on serum oestrogen levels on Day 4. However, the mice treated with anti-hCG did not litter; resorption of the embryos took place between Days 10 and 14 of pregnancy. Indirect immunofluorescence and quantitative immunoenzymic assays showed the presence of anti-ovine LH and anti-hCG reacting antigens in the mouse feto-placental unit. On Day 6, the values of reacting antigens (mean +/- s.d. absorbance units/10 micron section of embryo) were 0.050 +/- 0.002 with control globulins, 0.059 +/- 0.002 with anti-hCG-Ig and 0.196 +/- 0.018 with anti-LH-Ig; the corresponding values on Day 12 were 0.075 +/- 0.009, 0.402 +/- 0.02 and 0.416 +/- 0.015. The quantitative disposition of the reacting antigens to the two types of anti-gonadotrophins seems to bear a temporal relationship to their respective antifertility action. The pregnancy terminating action of immunoglobulins to ovine LH (Days 6, 7 & 8) and hCG (Days 8, 9 & 10) was counteracted by administration of 2 mg medroxyprogesterone acetate on Days 6, 9 and 12, indicating the importance of progesterone in the maintenance of pregnancy in the mouse.

Kainic acid lesion of the medial preoptic area resulted in the impairment of the thermoregulation and disruption of the reproductive cycle in female rats. The preoptic-anterior hypothalamic area from fetal donor was transplanted into the lesioned area ofthe host. After transplantation, these animals showed signs of estrus cyclicity and a recovery in basal temperature towards normal. They also showed a reduced shift of body temperature on exposure to a hot environment. Thus the female rats, which received transplantation, showed some degree of recovery of functions which were disrupted by kainic acid lesions.

Changes in the ependyma and periventricular brain tissues of the lateral, 3rd and 4th ventricles and the cervical spinal canal were studied electron-microscopically in young rabbits on the 9th day after injecting kaolin into the cisterna magna. The ependyma of the lateral ventricle overlying the white matter was notably stretched causing increased egress of CSF and disorganisation of the normal architecture of the white matter and capillaries. The neurons and glial cells close to the white matter showed edematous changes. The changes in the ependymal lining and the underlying grey matter were less severe in the dorsal part of the 3rd and the 4th ventricle. The ventral part of the 3rd ventricle was the least affected. The height and the arrangement of the ependymal cells, the surrounding grey matter with narrow interstitial spaces and the absorbing tanycytes seemed to be factors which were responsible for the minimal changes in these regions. The changes appeared to be reversible if the CSF pressure was relieved at this stage. The spinal canal remained unaffected in the majority of our hydrocephalic animals, which could probably be attributed to the type of animal and the degree of hydrocephalus.