Patterns of expression of the JIM4 arabinogalactan-protein epitope in cell cultures and during somatic embryogenesis in Daucus carota L.

Spatiotemporal patterns of expression of the cell-surface arabinogalactan-protein epitope defined by monoclonal antibody JIM4 (J.P. Knox et al., 1989, Development 106, 47–56) have been characterized by indirect immunofluorescence during the process of somatic embryogenesis in Daucus carota L. The JIM 4 epitope (J4e) occurred on cells established in culture from hypocotyl explants which appeared to derive, at least in part, from the epidermal cells of the hypocotyl. Cultures maintained in the presence of 2,4-dichlorophenoxyacetic acid developed proembryogenic masses of which only infrequent cells at the surface expressed J4e. Sub-culture at a low cell density and withdrawl of the synthetic auxin resulted in an increase in J4e expression in most surface cells and most abundantly in surface layers of cells at the future shoot end of developing embryos. The transition to heart-shaped embryos occurred concurrently with the expression of J4e by groups of cells beneath the developing cotyledons, at the junction of the future root and shoot. At this stage, J4e was also expressed by a single well-defined layer of cells at the surface of the embryos. Advancement to the mature torpedo stage was accompanied by the expression of the epitope on cells forming two regions of the future stele and of cells associated with the cotyledonary provascular tissue characteristic of the carrot seedling. At this stage there was substantially less expression of the marker antigen by epidermal cells, although infrequent expression by isolated cells of the epidermis was maintained. The correlation of J4e expression with the development and distinction of plant tissue patterns during somatic embryogenesis indicates a role for plasma-membrane arabinogalactan proteins in these processes.Abbreviations AGP arabinogalactan protein - 2,4-D 2,4-di-chlorophenoxyacetic acid - J4e JIM 4 epitope - PEM proembryogenic mass We thank Andrew Davis for photographic assistance and Roger Pennell for useful discussions.     

Developmental toxicity of soman in rats and rabbits

Soman (GD; phosphonofluoridic acid, methyl-,1,2,2-trimethylpropyl ester) is an organophosphate compound with potent anticholinesterase activity. To determine developmental toxicity, soman was administered orally to CD rats on days 6 through 15 of gestation at dose levels of 0, 37.5, 75, 150, or 165 micrograms/kg/day and to New Zealand White (NZW) rabbits on days 6 through 19 of gestation at dose levels of 0, 2.5, 5, 10, or 15 micrograms/kg/day. At sacrifice, gravid uteri were weighed and examined for number and status of implants. Individual fetal body weights and external, visceral, and skeletal malformations were recorded. Mean maternal weight changes, fetal implantation status/litter, fetal weight, and fetal malformations/litter were compared between dose groups. Monitors for maternal toxicity were net body weight change, treatment weight change, mortality, and clinical signs of toxicity such as lethargy, ataxia, and tremors. Maternal rats and rabbits in the high-dose groups exhibited statistically significant increases in toxicity and mortality when compared to controls. There were no significant dose-related effects among dose groups in the prevalence of postimplantation loss, malformations, or in average body weight of live fetuses per litter. There was no evidence of increased prenatal mortality or fetal toxicity in the CD rat or NZW rabbit following exposure to soman, even at a dose that produced significant maternal toxicity.     

Evaluation of two unstructured mathematical models for the penicillin G fedbatch fermentation

The mathematical model for the penicillin G fed-batch fermentation proposed by Heijnen et al. (1979) is compared with the model of Bajpai & Reuß (1980). Although the general structure of these models is similar, the difference in metabolic assumptions and specific growth and production kinetics results in a completely different behaviour towards product optimization. A detailed analysis of both models reveals some physical and biochemical shortcomings. It is shown that it is impossible to make a reliable estimation of the model parameters, only using experimental data of simple constant glucose feed rate fermentations with low initial substrate amount. However, it is demonstrated that some model parameters might be key factors in concluding whether or not altering the substrate feeding strategy has an important influence on the final amount of product.It is illustrated that feeding strategy optimization studies can be a tool in designing experiments for parameter estimation purposes.     

Localization of constitutive heat shock proteins in developing ascidians

Heat shock proteins (HSP) are a group of highly conserved proteins that regulate protein folding and ameliorate the effects of environmental stress. In the present study, the question of whether or not ascidian oocytes, embryos and larvae constitutively synthesize HSP was studied using HSP 60 and HSP 70 antibodies. Developmental stages obtained from Boltenia villosa, Cnemidocarpa finmarkiensis, Styela montereyensis and Corella willmeriana were examined for HSP using indirect immunocytochemistry. Myoplasm in oocytes and unfertilized eggs reacted with HSP 60 and 70 antibodies. HSP signals dramatically moved into the vegetal egg cytoplasm during ooplasmic segregation and colocalized with the myoplasm. In cleavage-stage embryos, HSP signals were partitioned with the myoplasm into muscle progenitor blastomeres and HSP signals were evident in the tail muscle cells of larvae. Immunoblots of proteins extracted from oocytes, eggs, embryos and larvae indicate that anti-HSP 60 recognizes a single band having an estimated molecular weight of 60 kDa. Egg centrifugation experiments suggest that most of the ascidian myoplasmic HSP are mitochondrial proteins. These results raise an intriguing possibility that mitochondria associated with the myoplasm perform biochemical functions that are unique to the embryonic muscle cell lineage.     

The 21-aminosteroid U-74389F increases the number of glial fibrillary acidic protein-expressing astrocytes in the spinal cord of control and wobbler mice

Summary 1. Wobbler mice suffer an autosomal recessive mutation producing severe motoneuron degeneration and dense astrogliosis, with increased levels of glial fibrillary acidic protein (GFAP) in the spinal cord and brain stem. They have been considered animal models of amyotrophic lateral sclerosis and infantile spinal muscular atrophy. 2. Using Wobbler mice and normal littermates, we investigated the effects of the membrane-active steroid Lazaroid U-74389F on the number of GFAP-expressing astrocytes and glucocorticoid receptors (GR). Lazaroids are inhibitors of oxygen radical-induced lipid peroxidation, and proved beneficial in cases of CNS injury and ischemia. 3. Four days after pellet implantation of U-74389F into Wobbler mice, hyperplasia and hypertophy of GFAP-expressing astrocytes were apparent in the spinal cord ventral and dorsal horn, areas showing already intense astrogliosis in untreated Wobbler mice. In control mice, U-74389F also produced astrocyte hyperplasia and hypertophy in the dorsal horn and hyperplasia in the ventral-lateral funiculi of the cord. 4. Givenin vivo U-74389F did not change GR in spinal cord of Wobbler or control mice, in line with the concept that it is active in membranes but does not bind to GR. Besides, U-74390F did not compete for [³H]dexamethasone binding when addedin vitro. 5. The results suggest that stimulation of proliferation and size of GFAP-expressing astrocytes by U-74389F may be a novel mechanism of action of this compound. The Wobbler mouse may be a valuable animal model for further pharmacological testing of glucocorticoid and nonglucocorticoid steroids in neurodegenerative diseases.     

Expressed Peromyscus maniculatus (Pema) MHC class I genes: evolutionary implications and the identification of a gene encoding a Qa1-like antigen

To gain insight into the evolution of rodent major histocompatibility complex (MHC) class I genes and identify important (conserved) nonclassical class I (class Ib) gene products and residues in these proteins, sixPeromyscus maniculatus MHC (Pema) class I cDNA clones were isolated and sequenced. FivePema class I cDNAs appeared most similar to mouse and rat classical class I (class Ia) genes. One exhibited highest similarity to anH2 class Ib gene,H2-T23 (encoding the Qa1 antigen). Phylogenetic trees constructed withPema, RT1, andH2 class I sequences suggested that the lineages of some rodent class Ib genes (e.g.,T23 andT24) originated prior toMus andPeromyscus speciation [>50 million years (My) ago]. Sequences of four Qa1-like proteins from three species permitted the identification of ten Qa1-specific amino acids. On the basis of molecular modeling, three residues showed the potential to interact with T-cell receptors and three residues (all corresponding to polymorphic positions among H2 class Ia proteins) were predicted to influence antigen binding. The recognition of mouse Qa1 proteins by a subset of T-cells in influenced by a locus,Qdm, which encodes the H2-D leader peptide. One of thePema class I cDNA clones classified asH2-K, D/L-like (class Ia) is predicted to encode an identical peptide, implying that an antigen binding protein (Qa1) and the antigen to which it binds (the product ofQdm) has been conserved for over 50 My. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U12822 (Pm13), U12885 (Pm41), U12886 (Pm52), U12887 (Pm62), U16846 (Pm11), and U16847 (Pm53)     

Urinary 6-Sulfatoxymelatonin Cycle-To-Cycle Variability

For either clinical or research purposes, the timing of the nocturnal onset in production of the urinary melatonin metabolite 6-sulfatoxymelatonin (UaMT6s-onset), has been proposed as a reliable and robust marker of circa-dian phase. However, given that most circadian rhythms show cycle-to-cycle variability, the statistical reliability of phase estimates obtained from a single study using UaMT6s-onset remains to be determined. Following 2 weeks of sleep diary and wrist actigraphy, 15 young, healthy good sleepers participated in four UaMT6s sampling sessions spaced 1 day apart. During the sampling sessions subjects remained indoors under low light conditions and hourly urine samples were collected from 19:00 to 02:00 h. Samples were subsequently assayed for UaMT6s using standard radioimmunographic techniques. UaMT6s-onset was determined by the time at which melatonin production exceeded the average of three proceeding trials by 100%. Sleep onset times were derived from sleep diary and actigraphic measures taken before the melatonin collection nights. We found that there was no significant variation between nights in group mean UaMT6s-onset times, and intraindividual variability was small. In addition, UaMT6s-onset times were highly and significantly correlated between nights (grand mean r = 0.804). Our results suggest that within 95% confidence interval limits, individual UaMT6s-onset estimates obtained from a single night UaMT6s-onset study can be used to predict subsequent UaMT6s-onset times within ±97 min. A close temporal relationship was also found between the timing of UaMT6s-onset and sleep onset. Overall, our results suggest that under entrained conditions single-session UaMT6s-onset studies can provide reliable individual UaMT6s-onset phase estimates and that the protocol described in this study is a practical and noninvasive methodology. (Chronobiology International, 13(6), 411-421, 1996)     

The modulation of calcium currents by the activation of mGluRs

Glutamatergic transmission in the central nervous system (CNS) is mediated by ionotropic, ligand-gated receptors (iGluRs), and metabotropic receptors (mGluRs). mGluRs are coupled to GTP-binding regulatory proteins (G-proteins) and modulate different second messenger pathways. Multiple effects have been described following their activation; among others, regulation of fast synaptic transmission, changes in synaptic plasticity, and modification of the threshold for seizure generation. Some of the major roles played by the activation of mGluRs might depend on the modulation of high-voltage-activated (HVA) calcium (Ca²⁺) currents. Some HVA Ca²⁺ channels (N-, P-, and Q-type channels) are signaling components at most presynaptic active zones. Their mGluR-mediated inhibition reduces synaptic transmission. The interference, by agonists at mGluRs, on L-type channels might affect the repetitive neuronal firing behavior and the integration of complex events at the somatic level. In addition, the mGluR-mediated effects on voltagegated Ca²⁺ signals have been suggested to strongly influence neurotoxicity. Rather different coupling mechanisms underlie the relation between mGluRs and Ca²⁺ currents: Together with a fast, membrane-delimited mechanism of action, much slower responses, involving intracellular second messengers, have also been postulated. In the recent past, the relative paucity of selective agonists and antagonists for the different subclasses of mGluRs had hampered the clear definition of the roles of mGluRs in brain function. However, the recent availability of new pharmacological tools is promising to provide a better understanding of the neuronal functions related to different mGluR subtypes. The analysis of the mGluR-mediated modulation of Ca²⁺ conductances will probably offer new insights into the characterization of synaptic transmission and the development of neuroprotective agents.     

Electro-immunoblotting characterization ofPseudo-nitzschia multiseries andP. pungens antigens recognized by antibodies directed against whole cells

Separate polyclonal antibodies have previously been developed against the domoic-acid-producingPseudonitzschia multiseries (=Pseudo-nitzschia pungens f.multiseries) and the non-toxicP. pungens (=P. pungens f.pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared againstP. multiseries strongly labelled the high molecular-weight antigens of allP. multiseries strains tested and showed little reactivity towardsP. pungens extracts on Western blots.P. pungens antibodies strongly labelled high molecular-weightP. pungens antigens and faintly labelled a fewP. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxicP. multiseries and non-toxicP. pungens are high molecular-weight (°>100kDa) polysaccharides located on the surface of these diatoms.Author for correspondence