Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Microsomal-catalyzed hydroperoxide-dependent C-oxidation of amines

Organic hydroperoxides are capable of supporting the C-oxidation of several different amines in the presence of hepatic microsomes. Evidence is presented that indicates that microsomal cytochrome P-450 acts as the catalyst. Removal of the NADPH-cytochrome $\text{c}$ oxidoreductase or essential phospholipid from microsomes does not significantly affect the peroxidase activity. Of the amine substrates C-oxidized by organic hydroperoxides in the presence of microsomes, only aminopyrine and dimethylaniline are rapidly oxidized by hydroperoxides in the presence of catalase. The catalase-mediated reaction can also be distinguished from the microsomal-catalyzed reaction by the use of differential inhibitors.     

Water chemistry of Lake Ladoga and Russian-Finnish intercalibration of analyses

As part of the Russian-Finnish research studies on Lake Ladoga, joint expeditions were organized in 1992 and 1993. Water samples were collected for intercalibration of chemical analysis methods and to monitor the chemical quality of the lake water.In August of 1992 water samples were taken from northern Lake Ladoga for intercalibration of Russian and Finnish analysis methods. In August 1993 water samples were collected from 23 sampling stations in all parts of the lake; some of these were also used for intercalibration purposes.The oxygen, colour and COD_Mn results were at the same level in the intercalibration. In 1993, the P_tot results obtained were acceptable. In N_tot, Fe and Mn analysis there seemed to be systematic and random errors between some results.The Secchi depth ranged from 1.5 m to 3.3 m. The average concentrations for the total phosphorus ranged from 15 µg 1^–1 to 29 µg 1^–1. The total nitrogen values were from 620 µg 1^–1 to 690 µg 1^–1. The N:P ratio varied from 24 to 40. The concentration of phosphorus indicated mesotrophic or even eutrophic conditions in the lake. Phosphorus seemed to be the limiting nutrient to bacteria and algae.     

Zooplankton of Lake Peipsi-Pihkva in 1909–1987

164 taxa were identified in the net zooplankton of the pelagial of L. Peipsi-Pihkva in 1909–1987, including 3 species of protozoans, 74 species of rotifers, 58 species of cladocerans, 28 species of copepods and 1 mollusc. One rotifer species, Ploesoma peipsiense Mäemets et Kutikova, has been described as new for science here. The zooplankton of L. Peipsi-Pihkva is remarkably rich in species including rarities in Estonia: Limnosida frontosa, Drepanothrix dentata, Bythotrephes longimanus, B. cederstroemi etc. Due to its large surface area, L. Peipsi-Pihkva provides a large scale of biotopes of a diverse trophic state and humic content, which support species with different ecological requirements. Most of the aquatory of the lake has lately been mesotrophic, favouring the coexistence of indicators of oligo- and mesotrophic state and species preferring a higher trophic state. The occurrece of 10 species of the genus Bosmina including B. berolinensis, B. gibbera, B. lilljeborgi, B. thersites and B. crassicornis, sparse in Estonian lakes, is the most noteworthy feature of the zooplankton of L. Peipsi-Pihkva. The coexistence of B. coregoni and B. berolinensis, B. gibbera, B. lilljeborgi etc. which were earlier regarded as subspecies of B. c. coregoni proves that they are different species producing usually no hybrids. The species composition was subjected to certain changes during the years under consideration. Larvae of Dreissena were first found in zooplankton in 1962. The oligo-mesotrophic indicator Holopedium gibberum occurred in the lake in 1909–1964, but was lacking in later samples.     

Developmental and Regional Expression of NMDA Receptor Subtypes Containing the NR2D Subunit in Rat Brain

Abstract: The regional and developmental expression of NMDA receptors containing the NR2D subunit was analyzed on the level of the subunit mRNA and protein in rat brain. RNase protection experiments indicated that among two proposed splice variants of the NR2D subunit, only the NR2D-2 subunit is expressed. The regional distribution of the NR2D subunit protein was visualized with a newly developed NR2D-2 subunit-specific antiserum on brain sections using the histoblot technique. In adult brain, NR2D immunoreactivity was mainly restricted to diencephalic, mesencephalic, and brainstem structures. During postnatal development, the NR2D subunit was detected transiently in certain regions, such as the ventro-basal complex of the thalamus, hippocampus, inferior colliculus, and brainstem reticular formation, suggesting that NR2D subunit-containing receptors play a role in these brain areas only during development. The level of NR2D subunit mRNA and protein decreased during late postnatal development. However, significant levels of NR2D subunit mRNA and protein were present in adulthood, in particular, in the globus pallidus, thalamus, subthalamic nuclei, and superior colliculus. These results indicate a functional relevance for NMDA receptors containing the NR2D subunit in the developing and adult brain, although its expression in the adult brain is less prominent and restricted to a few brain areas.     

Phytoplankton pigments and adenosine triphosphate (ATP) in Lake Peipsi-Pihkva

The material for pigment analysis was collected 1–3 times a year from Lake Peipsi-Pihkva in 1983, 1987, 1988, 1991 and 1992–1995. Concentrations of chlorophyll a, b and c (Chla, Chlb, Chlc), pheopigment (Pheo) and adenosine triphosphate (ATP) were measured biweekly in 1985–1986. The mean of all Chla values was 20.2 mg m^–1 (median 13.3 mg m^–1) indicating the eutrophic state of the lake. Average Chlb, Chlc, Pheo and carotenoid (Car) contents were 3.7 mg m^–3, 4.1 mg m^–3, 3.0 mg m^–3 and 4.8 mg m^–3, respectively. The average Chlb/Chla ratio was 22.9%, Chlc/Chla 23.4%, Pheo/Chla 38%, Car/Chla 37% and ATP/Chla 3%, the medians being 14.3, 13.6, 17.5, 39.4 and 1.9%, respectively. The proportion of Chla in phytoplankton biomass was 0.41%, median 0.32%. There were no significant differences in temperature, oxygen concentration, Chla, and ATP between the surface and bottom water; the lake was polymictic during the vegetation period. The Chla concentration had its first peak in May followed by a decrease in June and July. In late summer Chla increased again achieving its seasonal maximum in late autumn. The ATP concentration was the highest during spring and early summer, decreasing drastically in autumn together with the decline of primary production. ATP/Chla was the highest during the clear water period in June and early July, which coincided also with the high proportion of Chla in phytoplankton biomass. The highest Chla occurred in November (average 37.2 mg m^–3) when Secchi transparency was the lowest (1.05 m). Concentrations of Chlb, Chlc and carotenoids were the highest in August, that of Pheo in June. Concentrations of Chla and other pigments were the lowest in the northern part of Lake Peipsi (mean 14.7 mg m^–3, median 12.5 mg m^–3) and the highest in the southern part of Lake Pihkva (mean 47.9 mg m^–3, median 16.3 mg m^–3). An increase of Chla and decrease of Secchi depth could be noticed in 1983–1988, while in 1988–1994 the tendency was opposite.     

Continuous,high capacity reconstitution of nutrient media from concentrated intermediates

We designed an Integrated Media Preparation System (IMPS) for continuous, on-line preparation of cell culture media and delivery to intermediate storage vessels or directly to a bioreactor. Key components of the IMPS include: a high precision, continuous fluid mixing device; formulation-specific liquid medium concentrates; validated process controls and membrane filtration; and automated dispensing into large volume flexible plastic containers. The IMPS system is designed to produce sterile, single-strength liquid medium from common raw materials at a delivery rate of 1000–3000 liters per hour and will manufacture homogenous batches from several thousand liters to over 60,000 liters. Fortified nutrient media prepared from multi-component 50X concentrates have been demonstrated to accelerate bioreactor seed chains, increase product yield, and reduce the overall manufacturing cost of nutrient medium. A productivity matrix will analyze the fully-loaded costs and contrast alternative methods for media preparation against projected biological yield.Abbreviations IMPS Integrated Media Preparation System - 50X Nutrient fluid components formulated at fifty-fold final use concentration - 1X Nutrient fluid formulated at final, single-strength use concentration - cGMP Current Good Manufacturing Practices - SCADA Supervisory Control and Data Acquisition - PLC Process Logic Controller - LTI Life Technologies, Inc. - WFI Water for Injection - CIP Clean in place - SIP Sterilize in place - HPLC High performance liquid chromatography - DMEM Dulbecco's Modified Eagle's Medium     

Characterization and co-culture of novel nontransformed cell lines derived from rat endometrial epithelium and stroma

Summary Normal and neoplastic growth of epithelial cells depends on mutual interactions between epithelial and stromal cells. As a tool for the study of the underlying molecular mechanisms, we have developed temperature-sensitive, nontransformed cell lines derived from rat uterine epithelium and stroma by transfecting primary cultures with a temperature-sensitive mutant of the SV40 large T antigen. The epithelial and stromal cell lines obtained shared relevant morphological characteristics with the primary cells from which they were derived. Immunocytochemical analysis showed that the epithelial cell lines expressed the intermediate filament cytokeratin, whereas the stromal lines expressed the intermediate filament vimentin. Alkaline phosphatase activity was present in all cell lines examined. All cell lines were anchorage dependent and did not form foci. One epithelial cell line expressed oxytocin mRNA, a gene product recently shown to be highly expressed in vivo in the uterine epithelium at term. If grown on Matrigel, this cell line formed domelike structures, a further characteristic of its differentiated phenotype. In an attempt to reconstitute an endometrium in vitro, epithelial cells were seeded on top of a layer of stromal cells. Paraffin cross sections showed that this in vitro system consisted of a bilayer structure. Four to five cuboidal epithelial cells were typically anchored atop one stromal cell, forming an endometriumlike tissue. The present in vitro system should provide a useful model for further studies on endometrial functions and epithelial/stromal cell interactions at a molecular level.     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.     

Autoradiographic Distribution and Characteristics of High- and Low-Affinity Polyamine-Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin

Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [³H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [³H]ifenprodil (40 nM) in a biphasic manner with IC₅₀ values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC₅₀ values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [³H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [³H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [³H]ifenprodil sites, defined either by ifenprodil displacement curves or by [³H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [³H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [³H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC₅₀ of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [³H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [³H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [³H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.