Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

Modelling enteric bacteria survival in aquatic systems

We measured the uptake of carbon and inorganic nitrogen in nutrient-enriched water samples during 15-days incubation in summer in Lake Nakanuma, Japan. We calculated daily variations in neutral sugars and amino acids of the phytoplankton and estimated the efficiency of the increases in sugars and amino acids relative to photosynthetic activities. Only a small portion of carbon incorporated via photosynthesis was used for synthesis of sugars and amino acids during the incubation periods. The percentage increase in neutral sugars plus amino acids compared to photosynthetic rates ranged from 3.7 to 26.9% with an average of 12.8%. These findings suggest that large amounts of photosynthates were not used for the synthesis of cell components of phytoplankton and were lost through processes such as excretion and respiration.     

Distribution and production ofChironomus salinarius (Diptera: Chironomidae) in a shallow coastal lagoon in the Bay of Cádiz

The abundance, generation time and production ofChironomus salinarius larvae in a lagoon fish-pond system in the Bay of Cádiz were studied by taking monthly samples at 3 sites during 1991 and 1992. Numerical abundance and biomass of larvae showed considerable spatial, seasonal and interannual variation (ANCOVAs,P<0.001). The maximum mean annual density was 7048 larvae m^–2, and corresponded to a biomass of 3.08 g dry weight (DW) m^–2. It was recorded at the site with the lowest rate of water renewal. Seasonal patterns were similar at all sites, with main annual peaks of abundance and biomass in autumn-early winter. Chironomid density was positively related to the biomass of benthic macroalgae (P<0.001). The population studied was multivoltine with a probable average of five generations per year, with overlapping cohorts and a predominance of third- and fourth-instar larvae. Estimates of annual production ranged between 72.2 g DW m^–2 yr^–1 at the site with the lowest rate of water renewal in 1991 and 0.1 g DW m^–2 yr^–1 at the site with the highest rate of water renewal in 1992. Mean annual production and the production/biomass ratio for the system was estimated to be 16.8 g DW m^–2 yr^–1 and 12.7, respectively. Possible factors leading to the observed density fluctuations are discussed, as well as possible sources of error in production estimates.     

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions

In order to elucidate the relationship between hypertension and hypertrophy in the production of heat shock proteins, we studied the induction of the HSP72 synthesis by the heart and gracilis muscles of normo (WKY) and hypertensive (SHR) rats subjected to hyperthermia (42°C±0.5 for 15 min). Two age groups were investigated in each strain: young (2 months, with developing cardiac hypertrophy) and old (18 months, with fully developed chronic cardiac hypertrophy). The gracilis muscle never developed hypertrophy, independently of hypertension or aging. 72 kDa inducible protein was determined by Western blot analysis using a specific monoclonal antibody. We also used a commercial standard, loaded on each blot, to quantitate densitometrically the signal.The heart of young SHR responds to heat shock more than their normotensive age-matched control (298.8±24.7% vs 88.3 ±8.5%, p<0.001). This response is not maintained during aging as we did not find any significant difference between normo-and hypertensive old rats after exposure to hyperthermia (43.6±5.3% vs 65.3±10.4%).Unlike the heart, the gracilis muscle shows a basal spontaneous HSP72 synthesis in both the SHR (71.4±10.8%) and WKY (40.6±11.7%) animals. There was a significant increase in HSP72 synthesis in the gracilis muscle of young SHR with respect to their control (186.2±18.7% vs 115.8±9.9%, p<0.02) which was maintained also during aging (171.9±17.3% vs 95.2±10.5%, p<0.01).In conclusion, these data show that hypertension results in an increased synthesis of HSP72 both in cardiac and gracilis muscle in response to heat shock. This abnormal response is attenuated by aging in the heart but not in the gracilis muscle. Thus, the abnormality seems to be independent from hypertrophy and linked to genetic determination of the disease.     

Lignin and manganese peroxidase activity in extracts from straw solid substrate fermentations

Manganese and lignin peroxidase (MnP, LiP) activities were measured in straw extracts from cultures of Phanerochaete chrysosporium. Out of six MnP substrates, the MBTH/DMAB (3-methyl-2-benzothiazolinone hydrazone/3-(dimethylamino)benzoic acid), gave the highest MnP activity. Detection of LiP activity as veratryl alcohol oxidation was inhibited by phenols in the straw culture extracts. Appropriate levels of veratryl alcohol and peroxide (4 mM and 0.4 mM, respectively), and a restricted sample volume (not larger than 10%) were necessary to detect activity.     

Noradrenergic and adrenergic systems in the brain of the urodele amphibian,Pleurodeles waltlii,as revealed by immunohistochemical methods

The distribution of noradrenaline and adrenaline in the brain of the urodele amphibian Pleurodeles waltlii has been studied with antibodies raised against noradrenaline and the enzymes dopamine--hydroxylase and phenylethanolamine-N-methyltransferase. Noradrenaline-containing cell bodies were found in the anterior preoptic area, the hypothalamic nucleus of the periventricular organ, the locus coeruleus and in the solitary tract/area postrema complex at the level of the obex. Noradrenergic fibers are widely distributed throughout the brain innervating particularly the ventrolateral forebrain, the medial amygdala, the lateral part of the posterior tubercle, the parabrachial region and the ventrolateral rhombencephalic tegmentum. Putative adrenergic cell bodies were found immediately rostral to the obex, ventral to the solitary tract. Whereas the cell bodies and their dendrites were Golgi-like stained, axons were more difficult to trace. Nevertheless, some weakly immunoreactive fibers could be traced to the basal forebrain. A comparison of these results with data previously obtained in anurans reveals not only several general features, but also some remarkable species differences.Abbreviations Acc Nucleus accumbens - AP area postrema - Apl amygdala, pars lateralis - Apm amygdala, pars medialis - ca commissura anterior - Cb cerebellum - cc central canal - Dp dorsal pallium - epl external plexiform layer - gl glomerular layer of the olfactory bulb - H ganglion habenulae - igl internal granular layer - Ip nucleus interpeduncularis - Lc locus coeruleus - Ll lateral line lobe - Lp lateral pallium - Ls lateral septum - ml mitral cell layer - Mp medial pallium - Ms medial septum - nPT nucleus pretectalis - NPv nucleus of the periventricular organ - nV nervus trigeminus - oc optic chiasm - Poa preoptic area - Ri nucleus reticularis inferior - SC nucleus suprachiasmaticus - sol solitary tract - Str striatum - thd thalamus dorsalis - thv thalamus ventralis - To tectum opticum - TP tuberculum posterius - V ventricle - VH ventral hypothalamic nucleus - III nucleus nervi oculomotorii - IXm nucleus motorius nervi glossopharyngei - Xm nucleus motorius nervi vagi     

Inhibition by Cl− channel blockers of the volume-activated,diffusional mechanism of inositol transport in primary astrocytes in culture

[³H]Inositol accumulated by rat brain cultured astrocytes is released when cells swell by exposure to solutions of decreased osmolarity. Activation of inositol efflux was proportional to reductions in osmolarity from 30%–70%. This volume-activated inositol efflux pathway was increased (27%) in Na⁺-free medium and decreased (22%) in Cl^–-free medium. It was independent of extracellular Ca²⁺ and was reduced (30%) in the presence of the intracellular chelator [1,2-bis(o-aminophenoxy) ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester] (BAPTA-AM). The inositol efflux pathway was markedly inhibited by Cl^– channel blockers, which at maximal inhibitory concentrations decreased inositol efflux by 70%–83%. The potency range of the drugs was: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)>1–9, dideoxyforskolin>4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS)>niflumic acid. Inositol efflux was strongly inhibited by the SH blocker N-ethyl maleimide (NEM), which at 100 M abolished inositol release. Inositol efflux can be reversed by increasing its extracellular concentration, suggesting that the efflux is mediated by a diffusional pathway whose direction is given by the concentration gradient. The inhibition of volume-associated fluxes of inositol by Cl^– channel blockers supports the suggestion of an anion channel as the common pathway for inorganic and organic osmolytes in cultured astrocytes.     

Constrained C-terminal hexapeptide neurotensin analogues containing a 3-oxoindolizidine skeleton

Summary In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT_8–13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro¹⁰-Tyr¹¹ fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III -turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT_8–13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala¹¹]-NT_8–13 and only five- to ninefold lower than that of the corresponding analogue, [Phe¹¹]NT_8–13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT_8–13 analogues and [Phe¹¹]NT_8–13 or NT_8–13, it is not clear whether the -turn around Pro¹⁰-AA¹¹ (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro¹⁰-Tyr¹¹ dipeptide in NT_8–13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors.     

Solid-state fermentation of sugarcane bagasse with Flammulina velutipes and Trametes versicolor

The mushroom Flammulina velutipes and the white-rot fungus Trametes versicolor were cultivated separately on sugarcane bagasse for 40 days. Trametes versicolor produced laccase and manganese-peroxidase activities, showing a simultaneous degradation of lignin and holocellulose. However, only phenoloxidase activity was found with Flammulina velutipes. A preferential degradation of lignin was detected in F. velutipes, which exhibited a greater reduction in the ratio of weight loss to lignin loss than T. versicolor. A decrease in the syringyl/guaiacyl ratio observed with both fungi indicated the preferential degradation of non-condensed (syringyl-type) lignin units. An increase in the relative abundance of aromatic carboxylic acids suggested that the oxidative transformation of lignin unit side-chains was occurring. This was more noticeable with Flammulina velutipes than with T. versicolor.     

A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis

Bacillus thuringiensis var. israelensis (BTI), serotype 14, which produces parasporal crystals toxic to certain dipteran larvae, was analyzed by agarose gel electrophoresis and found to contain a complex plasmid array. Eight plasmids were detected, with approximate sizes of 3.3, 4.2, 4.9, 10.6, 68, 75, 105, and 135 MDa, as well as a plasmidlike linear DNA element of ~10 MDa. Partially cured mutants of BTI implicated the 75-MDa plasmid in crystal production. Fifteen independently isolated acrystalliferous (Cry^?) mutants were found to lack this plasmid. In plasmid transfer experiments, several of the BTI plasmids transferred into a plasmid-free, Cry^? BTI recipient, but only transfer of the 75-MDa plasmid converted the recipient to crystal toxin production. The presence or absence of mosquito-toxic activity in all Cry⁺ and Cry^? variants of BTI was confirmed by bioassay of sporulated cultures against larvae of Aedes aegypti. Southern blot analyses revealed that in one unusual Cry⁺ variant in which no 75-MDa plasmid band was detectable, plasmid sequences were still present, possibly integrated into the chromosome. The 75-MDa plasmid could also apparently recombine with the 68-MDa plasmid, to which it was partially homologous.