EFFECTS OF CELL DENSITY,TEMPERATURE, AND LIGHT INTENSITY ON GROWTH AND STALK PRODUCTION IN THE BIOFOULING DIATOM ACHNANTHES LONGIPES (BACILLARIOPHYCEAE)1

Achnanthes longipes Ag. is a marine stalk‐forming diatom that grows in dense biofilms. The effects of cell density, temperature, and light on growth and stalk production were examined in the laboratory to determine how they affected the ability of this diatom to form a biofilm. Stalk production abruptly increased when A. longipes was cultured at a density of 5.4 × 10³ cells·mL ^{? 1} 1 Received 23 February 2002. Accepted 22 July 2002.
, with a lag before stalk production occurring in cultures initiated at lower densities. Growth occurred at all temperatures from 8 to 32° C, with maximum growth at 26° C. Growth rate was light saturated at 60 μmol photons·m ^{? 2}·s ^{? 1} 1 Received 23 February 2002. Accepted 22 July 2002.
. Stalk production was determined as the proportion of cells producing stalks and stalk length in response to various temperatures and light intensities at high (5000 cells·mL ^{? 1} 1 Received 23 February 2002. Accepted 22 July 2002.
) and low (500 cells·mL ^{? 1} 1 Received 23 February 2002. Accepted 22 July 2002.
) densities. More cells formed stalks at high density, with no difference in stalk length. The proportion of cells producing stalks was maximal at 20° C, with little change at 17–32° C. Stalk length was at a maximum between 14 and 26° C. Stalk production showed little change in response to varying light intensity. The results of an earlier investigation on the effects of bromide concentration on stalk formation were expressed as the proportion of cells forming stalks and the lengths of the stalks. Both measures of stalk production varied with bromide concentration, with maximum values at 30 mM bromide. The increased stalk production at higher densities may be a means of elevating cells above the substrate to avoid competition in the dense biofilm.     

INTERACTIVE EFFECTS OF PAR AND UV‐B RADIATION ON PSII ELECTRON TRANSPORT IN THE MARINE ALGA DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

To better understand the interactions between PAR and UV‐B radiation in microalgae, the marine chlorophyte alga Dunaliella tertiolecta was subjected to a UV‐B flux of 4.1 W·m ^{? 2} (unweighted) with varying PAR fluxes. Rate constants for damage and repair processes during UV‐B exposure increased with PAR flux. However, recovery after UV‐B exposure increased with PAR up to 300 μmol quanta·m ^{? 2}·s ^{? 1} 1 Received 17 September 2002. Accepted 19 February 2003. , beyond which photoinhibition of PSII electron transport was found to decrease recovery rates. In the absence of PAR during the post UV‐B exposure period, no recovery was seen, indicating that perhaps the lack of light available for photosynthesis depresses repair either directly or indirectly by affecting ATP synthesis. Possible mechanisms for the observed interactions between PAR and UV‐B exposure are discussed.     

INTERACTIONS BETWEEN NITRATE UPTAKE AND NITROGEN FIXATION IN CONTINUOUS CULTURES OF THE MARINE DIAZOTROPH TRICHODESMIUM (CYANOBACTERIA)1

Diazotrophic cyanobacteria can take up combined nitrogen (nitrate, ammonium, amino acids, dissolved organic nitrogen) from solution, but the interaction between N₂ fixation and uptake of combined nitrogen is not well understood. We studied the effects of combined nitrogen ) additions on N₂ fixation rates in the cyanobacterium Trichodesmium erythraeum (IMS‐101) maintained in continuous culture in an N‐free medium (YBCII) and a 12:12‐h light:dark cycle. We measured acetylene reduction rates, nutrient concentrations, and biomass throughout the 12 h of illumination after the addition of nitrate (0.5–20 μM) at the start of the light period. Compared with unamended controls, Trichodesmium showed strong inhibition of acetylene reduction (up to 70%) in the presence of , with apparent saturation of the inhibition effect at an initial concentration of approximately 10 μM. The inhibition of acetylene reduction persisted through much of the light period as concentration in the culture vessel decreased. Recovery of N₂ fixation was observed late in the light period in cultures amended with low concentrations of (<5 μM) when ambient concentrations had decreased to 0.3–0.4 μM in the culture vessel. Nitrate uptake accounted for as much as 86% of total N uptake and, at the higher treatment concentrations, more than made up for the observed decrease in N₂ fixation rates. We conclude that Trichodesmium can obtain significant quantities of N through uptake of nitrate and does so in preference to N₂ fixation when sufficient is available.     

A REVISED ESTIMATE OF THE IRON USE EFFICIENCY OF NITROGEN FIXATION,WITH SPECIAL REFERENCE TO THE MARINE CYANOBACTERIUM TRICHODESMIUM SPP. (CYANOPHYTA)1

Estimates of the iron use efficiency (IUE) for diazotrophic plant growth have been used to suggest iron limitation of marine N₂ fixation. However, in the course of these inferences, neither the physiological complexity of these estimates nor the specific physiological parameters of marine diazotrophs were evaluated. Here, a semiempirical prediction of the IUE of diazotrophic growth for Trichodesmium was computed from considerations of the Fe content and reaction rates of the nitrogenase complex and PSI:PSII ratios, as well as field measurements of Mehler activity, cellular Fe‐superoxide dismutase activity, and diel variability in C and N₂ fixation. With a PSI:PSII ratio of 1 and 48% Mehler activity, the instantaneous IUE (0.33 mol C fixed·mol cellular Fe ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. ·s ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. ) was only 4‐fold lower than that calculated for a phytoplankter growing on reduced N. We computed a range of daily integrated IUE values from 2900 to 7700 mol C·mol Fe ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. ·d ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. , accounting for the diel variability in C and N₂ fixation as well as the uncertainties in cyanobacterial nitrogenase biochemistry and PSI:II ratios of field‐collected Trichodesmium. The lowest observed Fe‐superoxide dismutase:C quota of 2.9 (μmol:mol) suggests a maintenance requirement for this enzyme. The maintenance Fe:C requirement of 13.5 μmol:mol (derived from cultures of Trichodesmium IMS 101) and values of the IUE yielded an Fe requirement ranging from 27 to 48 Fe:C (μmol:mol) to achieve a diazotrophic growth rate of 0.1 d ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. . Based on these predicted requirements, the Fe:C contents of Caribbean Sea and most North Atlantic Ocean populations sampled thus far exceed that required to support the observed rates of N₂ fixation.     

CONFIRMATION OF DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA AUSTRALIS (BACILLARIOPHYCEAE) ISOLATED FROM IRISH WATERS1

A nonaxenic isolate of the potentially toxic diatom Pseudo‐nitzschia australis (Frenguelli) from Irish waters was tested in two separate batch culture experiments. When grown under a low irradiance (～12 μmol photons·m ^{? 2}·s ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
; 16:8‐h light:dark cycle) for up to 40 days, the culture produced only trace amounts of the neurotoxin domoic acid (DA) during late stationary phase. Growth at a higher irradiance (～115 μmol photons·m ^{? 2}·s ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
; 12:12‐h light:dark cycle) resulted in DA production starting during late exponential phase and reaching a maximum concentration of 26 pg DA·cell ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
during late stationary phase. Liquid chromatography coupled to mass spectrometry was used to confirm the identity of DA in the culture. Irradiance and photoperiod could be important factors that contribute directly or indirectly to the control of DA production in P. australis. This is the first record of a DA‐producing diatom in Irish waters, and results indicate P. australis may have been the source of DA that has recently contaminated shellfisheries in this area.     

LACK OF CALBINDIN-D28K ALTERS RESPONSE OF THE MURINE CIRCADIAN CLOCK TO LIGHT

A strong stimulus adjusting the circadian clock to the prevailing light-dark cycle is light. However, the circadian clock is reset by light only at specific times of the day. The mechanisms mediating such gating of light input to the CNS are not well understood. There is evidence that Ca²⁺ ions play an important role in intracellular signaling mechanisms, including signaling cascades stimulated by light. Therefore, Ca²⁺ is hypothesized to play a role in the light-mediated resetting of the circadian clock. Calbindin-D28k (CB; gene symbol: Calb1) is a Ca²⁺ binding protein implicated in Ca²⁺ homeostasis and sensing. The absence of this protein influences Ca²⁺ buffering capacity of a cell, alters spatio-temporal aspects of intracellular Ca²⁺ signaling, and hence might alter transmission of light information to the circadian clock in neurons of the suprachiasmatic nuclei (SCN). We tested mice lacking a functional Calb1 gene (Calb1^?/?) and found an increased phase-delay response to light applied at circadian time (CT) 14 in these animals. This is accompanied by elevated induction of Per2 gene expression in the SCN. Period length and circadian rhythmicity were comparable between Calb1^?/? and wild-type animals. Our findings indicate an involvement of CB in the signaling pathway that modulates the behavioral and molecular response to light. (Author correspondence: urs.albrecht@unifr.ch)     

ISOLATION OF DI(HYDROXYMETHYL)DIHYDROXYPYRROLIDINE FROM THE CYANOBACTERIAL GENUS CYLINDROSPERMUM THAT EFFECTIVELY INHIBITS DIGESTIVE GLUCOSIDASES OF AQUATIC INSECTS AND CRUSTACEAN GRAZERS1

An effective glucosidase inhibitor was isolated from the cyanobacterial genus Cylindrospermum. Its chemical structure was determined by MS and NMR spectrometry to be di(hydroxymethyl)dihydroxypyrrolidine (DMDP; 2(R),5(R)‐bis‐(hydroxymethyl)‐3(R), 4(R)‐dihydroxypyrrolidine). Its identity was established by comparison with an authentic compound. All five species of Cylindrospermum investigated synthesized this compound but accumulated it to a different extent intracellularly. Particularly active producers were the axenic C. licheniforme (22 pmol·nmol chl a ^{? 1} 1 Received 5 March 2002. Accepted 2 October 2002. ) and a monoxenic unknown species of Cylindrospermum that contained the maximum amount (159 pmol·nmol chl a ^{? 1} 1 Received 5 March 2002. Accepted 2 October 2002. ). The major part of DMDP was found to be extracellular for all species investigated. The isolated compound inhibited digestive α‐ and β‐glucosidases isolated from crustacean zooplankton (IC₅₀ 19 and 49 nM, respectively). The bacterial 1‐deoxynojirimycin, which was used as a well‐studied reference glucosidase inhibitor, was less inhibitory (IC₅₀ 520 and 2190, respectively). Digestive enzymes of macrozoobenthos (chironomids, trichoptera, and ephemeroptera) were less sensitive to DMDP. The insect digestive β‐glucosidase was more effectively inhibited than the α‐glucosidase. Beside others, the ecological function of the glucosidase inhibitor may be the reduction of the digestibility of the cyanobacterium for grazers.     

THE EFFECT OF AGITATION AND TURBULENCE OF THE GROWTH MEDIUM ON THE GROWTH AND VIABILITY OF MICROCYSTIS

SUMMARY

Observation of natural blooms of Microcystis, suggested that increased turbulence plays a role in retarding bloom formation of Microcystis. In laboratory experiments the influence of turbulence mediated by a magnetic stirrer on the growth and viability of Microcystis in batch cultures was determined. The different turbulences (0, 25, 75, 126, 209 and 314 cm sec^?1 linear velocity) had no effect on the growth rate. There was a highly significant correlation between the linear velocity and percentage viability as determined by a plating and serial dilution method. The viability ranged from 0,8% for stationary cultures to 99,2% for vigorously stirred (314 cm sec^?1 linear velocity) cultures.     

Immunochemical ANALYSIS OF GLYCOSYLATED AND nonglycosylated dla class I antigens

Dog peripheral blood lymphocytes, when cultured with ³⁵S-methionine in the presence of tunicamycin, synthesize DLA molecules consisting of ₂-microglobulin and a heavy chain approximately 3000 daltons lower in apparent mol. wt. than observed in control cases. This difference in mol. wt. is consistent with the fact that a single N-linked carbohydrate side chain is present on the heavy chain of DLA class I antigens. There is no evidence of polymorphism in the DLA light chain ( ₂m). Both glycosylated and nonglycosylated forms of the heavy chain, however, show microheterogeneity, which can be related to tissue-type. Analysis by two-dimensional electrophoresis shows that the biochemical heterogeneity in the DLA heavy chain is less than expected from DLA serology, and less than found in HLA class I antigens. The data are consistent with the fact that the products of only a single DLA class I locus are detected.Abbreviations used in this paper ₂m beta-2-microglobulin - 2D two dimensional - DLA dog MHC - HLA human MHC - Ia I-region associated - MHC major histocompatibility complex - PBL peripheral blood lymphocytes - PHA-M phytohaemagglutinin-muco - pI isoelectric point - RLA rabbit MHC - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

THE COMPARTMENTATION OF GLUTAMATE AND ITS METABOLITES IN FRACTIONS OF NEURON CELL BODIES AND NEUROPIL; STUDIED BY INTRAVENTRICULAR INJECTION OF [U-14C]GLUTAMATE

Abstract—

• 1 The in vivo metabolism of glutamate in rat neuron cell bodies and neuropil was studied after intraventricular injection of (U-¹⁴C)glutamic acid followed by separation of the tissue into neuronal and neuropil fractions.

• 2 The losses of amino acid and of radioactivity during the fractionation were equivalent. Recoveries were: glutamate, 32; glutamine, 15; aspartate, 25; GABA, 41; alanine, 30 per cent. In the washed cell fractions glutamine was 45 per cent and alanine 132 per cent higher in the neuronal fraction, glutamate was 62, GABA 77 and aspartate 95 per cent of neuropil levels. This contrasted with results obtained previously for in vitro incorporation. Calculation from these results indicated that 28 per cent of the original cell suspension was neuronal, 72 per cent neuropil. In the final cell preparations, 29 per cent of the neuron cell bodies and 26 per cent of the neuropil were recovered.

• 3 Specific activity of glutamate in the neuronal fraction 15 min after injection was higher than in the original suspension, but had declined to 30 per cent of its initial value by 2 h. In the neuropil, specific activity of glutamate was below that of the cell suspension at 15 min, but at later times rose above it by up to 40 per cent.

• 4 Radioactivity was detected in aspartate and glutamine 15 min after injection and GABA by 60 min after injection. In the original cell suspension the specific activity of glutamine was higher than that of glutamate at all times (the Waelsch effect) but aspartate and GABA were lower than glutamate.

• 5 In the neuronal fraction the specific activity of glutamine was below that of glutamate at all times, indicating a precursor-product relationship. In the neuropil fraction, glutamine specific activity remained above glutamate for the first hour.

• 6 These results are discussed in relation to the interpretation of the Waelsch effect in terms of metabolic compartmentation.

     

In Vivo Mutational Analysis of YtvA from Bacillus subtilis: MECHANISM OF LIGHT ACTIVATION OF THE GENERAL STRESS RESPONSE

The general stress response of Bacillus subtilis can be activated by stimuli such as the addition of salt or ethanol and with blue light. In the latter response, YtvA activates σ^B through a cascade of Rsb proteins, organized in stressosomes. YtvA is composed of an N-terminal LOV (light, oxygen, and voltage) domain and a C-terminal STAS (sulfate transporter and anti-sigma factor) domain and shows light-modulated GTP binding in vitro. Here, we examine the mechanism of YtvA-mediated activation of σ^B in vivo with site-directed mutagenesis. Constitutive off and constitutive on mutations have been identified. Disruption of GTP binding in the STAS domain eliminates light activation of σ^B. In contrast, modification of sites relevant for phosphorylation of STAS domains does not affect the stress response significantly. The data obtained are integrated into a model for the structure of full-length YtvA, which presumably functions as a dimer.LOV² domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted Jα (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8, 9). Stress via the addition of salt or ethanol (for a review, see Ref. 10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13, 14). YtvA, which mediates light activation of σ^B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16).When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9, 14, 17, 18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating σ^B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of σ^B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180° upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central β-sheet and the C-terminal linker region, the Jα-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the σ^B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavin-binding pocket, the β-sheet of the LOV domain, and the GTP-binding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approach was used to model the structure of full-length YtvA. The model suggests that light modulates accessibility of the GTP-binding site of the STAS domain of YtvA.     

THE EFFECT OF SOFT SELECTION ON THE VARIABILITY OF A QUANTITATIVE TRAIT

The equilibrium phenotypic variance of a normally distributed quantitative character P under soft selection is studied. This character is assumed to undergo Gaussian stabilizing selection W(p, x) = exp[–(p – x)²/2w²]. The environmentally determined optimum (x) is a normal variable with variance s². A stable equilibrium with is found, so that increases both with increasing environmental heterogeneity and with increasing local intensity of stabilizing selection. It is shown that both genetic and environmental components of the variance are selected until this equilibrium is reached. Habitat selection, supposed to be normal (with variance H²) around the optimum, also increases the value. Nevertheless, relatively intense local stabilizing selection (w < s) and accurate habitat choice (H < s) are required for the initial spread and the evolutionary stability of this habitat selection.     

SEAWEED EXTRACTS AS A POTENTIAL TOOL FOR THE ATTENUATION OF OXIDATIVE DAMAGE IN OBESITY‐RELATED PATHOLOGIES1

Recent studies suggest that seaweed extracts are a significant source of bioactive compounds comparable to the dietary phytochemicals such as onion and tea extracts. The exploration of natural antioxidants that attenuate oxidative damage is important for developing strategies to treat obesity‐related pathologies. The objective of this study was to screen the effects of seaweed extracts of 49 species on adipocyte differentiation and reactive oxygen species (ROS) production during the adipogenesis in 3T3‐L1 adipocytes, and to investigate their total phenol contents and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging activities. Our results show that high total phenol contents were observed in the extracts of Ecklonia cava (see Table 1 for taxonomic authors) (681.1 ± 16.0 μg gallic acid equivalents [GAE] · g^?1), Dictyopteris undulata (641.3 ± 70.7 μg GAE · g^?1), and Laurencia intermedia (560.9 ± 48.1 μg GAE · g^?1). In addition, DPPH radical scavenging activities were markedly higher in Sargassum macrocarpum (60.2%), Polysiphonia morrowii (55.0%), and Ishige okamurae (52.9%) than those of other seaweed extracts (P < 0.05). Moreover, treatment with several seaweed extracts including D. undulata, Sargassum micracanthum, Chondrus ocellatus, Gelidium amansii, Gracilaria verrucosa, and Grateloupia lanceolata significantly inhibited adipocyte differentiation and ROS production during differentiation of 3T3‐L1 preadipocytes. Furthermore, the production of ROS was positively correlated with lipid accumulation (R² = 0.8149). According to these preliminary results, some of the seaweed extracts can inhibit ROS generation, which may protect against oxidative stress that is linked to obesity. Further studies are required to determine the molecular mechanism between the verified seaweeds and ROS, and the resulting effects on obesity.

Table 1. List of Korean seaweed extracts of 49 species evaluated in this experiment.

Effects of hydroxylamine and silicomolybdate on the decay in delayed light emission in the 6–100 μs range after a single 10 ns flash in pea thylakoids

Measurements are reported on μs delayed light emission, following a single 10 ns excitation flash, in Alaska pea thylakoids treated with hydroxylamine (NH₂OH) or with silicomolybdate.

1. In thylakoids treated with 2 mM NH₂OH in the light, or in the dark, the quantum yield of delayed light emission is considerably enhanced. A 10 μs lifetime component of delayed light emission is not significantly changed, whereas a 50–70 μs lifetime component is increased. MnCl₂ and diphenylcarbazide are unable to reverse the above effects of NH₂OH treatment. Thus Mn²⁺ and diphenylcarbazide must not donate electrons directly to reaction center II but on the oxygen-evolution side of the NH₂OH block.
2. When the closed form of photosystem II reaction centers (P₆₈₀Q^-), where P₆₈₀ is the reaction center chlorophyll and Q is a ‘stable’ electron acceptor, is generated by preillumination of NH₂OH-treated thylakoids with diuron present, the μs delayed light emission is inhibited, but a low level residual delayed light emission remains. Possible origins of this emission are discussed. It is believed that the best explanation for residual DLE is the existence of another acceptor besides Q that partakes in charge separation and rapid dissipative recombination when the reaction center is in the P₆₈₀Q^- state.
3. The quantum yield of delayed light emission from ‘closed’ reaction centers (P₆₈₀ ⁺Q^-) that have all charge stabilization reactions (i.e., flow of electrons to P₆₈₀ ⁺ and out of Q^-) blocked by NH₂OH treatment and addition of diuron is 1.1×10^-3 for components measured in a range from 6 to 400 μs and extrapolated to zero time.
4. The addition of silicomolybdate, which accepts electron from Q^-, causes delayed light emission in the μs range to be greatly inhibited.

     

NOVEL SYNTHESIS OF seco TYPE OF ACYCLO C-NUCLEOSIDES OF 1,2,4-TRIAZOLE AND 1,2,4-TRIAZOLO[3,4-b][1,3,4]THIADIAZINE

The seco C-nucleosides 3-(1,2,3,4,5-penta-O-acetyl-D-gluco- and D- galacto-pentitol-1-yl)-1H-1,2,4-triazoles (8 and 9) were obtained in a one pot by deamination and dethiolation of 4-amino-3-(D-gluco- and D-galacto-pentitol-1-yl)-5-mercapto-1,2,4-triazoles (1 and 2), respectively, using sodium nitrite in orthophosphoric acid and subsequent acetylation. Condensation of 1, 2, and 4-amino-3-(D-glycero-D-gulo-hexitol-1-yl)-5-mercapto-1,2,4-triazole (12) with phenacylbromide (11) afforded the corresponding 3-(D-gluco-, D-galacto-pentitol-1-yl) and 3-(D-glycero-D-gulo-hexitol-1-yl)-6-phenyl-7H-1,2,4- triazolo[3,4-b][1,3,4] thiadiazines (15, 16, and 17). Acetylation of 15–17 gave the penta- and hexa-O-acetyl derivatives 18–20, respectively. The structures were confirmed by using ¹H, ¹³C, and 2D NMR spectra, DQFCOSY, HMQC, and HMBC experiments. The favored conformational structures were deduced from the vicinal coupling constants of the protons.     

PHENYLALKYL BACKBONE MODIFIED OLIGODEOXYNUCLEOTIDES,THEIR SYNTHESIS AND THE INFLUENCE OF THE ALKYL CHAIN LENGTH

Phenylalkyl modified phosphoramidites (alkyl chain length n = 1,2,3,5; Fig. 1) were synthesised and incorporated into a DNA hexamer (5′-d(GCCp-GCG); p = place of modification). The obtained diastereomeres were separated by RP-HPLC. After hybridisation with the complementary DNA strand T_m-value and thermodynamic data were measured. The stability of duplexes depends on the linker length and the absolute configuration of the backbone modified oligodeoxynucleotides (Rp, Sp).

PHENYLALKYL BACKBONE MODIFIED OLIGODEOXYNUCLEOTIDES, THEIR SYNTHESIS AND THE INFLUENCE OF THE ALKYL CHAIN LENGTH

All authors

Stefan Amberg & Joachim W. Engels

https://doi.org/10.1081/NCN-100002535

Published online:

07 February 2007

Figure 1. Structure of Rp- and Sp-configurated oligomers; synthesised phosphoramidites.     

18.

THE RESPONSE OF PER1 TO LIGHT IN THE SUPRACHIASMATIC NUCLEUS OF THE DIURNAL DEGU (OCTODON DEGUS)     

《Chronobiology international》2013,30(6):1263-1271

Several studies suggest that the circadian systems of diurnal mammals respond differently to daytime light than those of nocturnal mammals. We hypothesized that the photosensitive “clock” gene Per1 would respond to light exposure during subjective day in the suprachiasmatic nucleus of the diurnal rodent, Octodon degus. Tissue was collected 1.5–2?h after a 30?min light pulse presented at five timepoints across the 24?h day and compared to controls maintained under conditions of constant darkness. Per1 mRNA was quantified using in situ hybridization. Results showed that the rhythmicity and photic responsiveness of Per1 in the degu resembles that of nocturnal animals. (Author correspondence: hagenaue@umich.edu)     

19.

CHANGES IN POLYSOMES OF THE DEVELOPING RAT BRAIN   总被引：1，自引：0，他引：1  

S. Yamagami  K. Mori 《Journal of neurochemistry》1970,17(6):721-731

Abstract— Rat brain polysomes were prepared from a deoxycholate-treated postmito-chondrial supernatant in the presence of 2% bentonite and 1 mg/ml of yeast RNA to prevent partial degradation during preparation.

• 1 The polysomal preparations had an absorption maximum at 260 mμ and an absorption minimum at 235 mμ. The ratio of absorption maximum to minimum and the RNA to protein ratio were 1·58 and 1·06 respectively in 6-day-old rat brain polysomes. The sedimentation patterns showed six distinct peaks with sedimentation coefficients of 235S, 185S, 173S, 135S, 100S and 80S, indicating that these preparations have the characteristics of pure heavy polysomes.

• 2 The rate of [¹⁴C]phenylalanine incorporation into brain polysomal protein was maximal at approximately 10 days of age and decreased thereafter. A similar progressive reduction with increasing age was found in the stimulation of phenylalanine incorporation by the addition of 60 μg/tube of polyuridylic acid. However, the incorporation of phenylalanine into young rat brain polysomes was usually greater even with the addition of polyuridylic acid than in the older animals.

• 3 The comparative studies on sucrose density gradient centrifugation of polysomes between young and adult rat brains showed a considerable decrease of heavy polysomes in the older animals.

• 4 The effect of various factors on the stability of brain polysomes from both ages has been studied. The rates of RNA, protein and acid-soluble phosphorus release from polysomes of the adult rat brains were usually greater in the presence of high salt concentration, ethylenediaminetetra-acetic acid and urea than those from the corresponding preparations of younger animals. On the basis of evidence obtained from the above results it suggested that the adult brain polysomes were more unstable than those of younger animals.

• 5 The amount of polysomal RNA linearly increased up to the first 20 days after birth and then levelled off. The ratio of G + C/A + U of polysomal RNA was less in the young rat brains, falling to 1·30 as compared to 1·50 in older animals. The differences were statistically significant at less than a 1% level of confidence.

• 6 Polysomal preparations also contained RNase, phosphomonoesterase, phospho-diesterase and 5′-nucleotidase activities which cannot be washed off. The specific activities of these enzymes were generally higher in young rat brains than those in the adult.

     

20.

Enzymatic Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48     

Masaru Matsuo  Tsuneo Yasui  Tatsuyoshi Kobayashi 《Bioscience, biotechnology, and biochemistry》2013,77(9):1601-1606

Some enzymatic properties of Malbranchea β-xylosidase were investigated. The β- xylosidase activity was inhibited by Hg²⁺, Zn²⁺, Cu²⁺, N-bromosuccinimide, p-chloromercuribenzoate and sodium laurylsulfate, while this activity was activated by Ca²⁺. The enzyme released xylose as the end product even from 10% xylobiose solution without forming any xylooligosaccharides. The enzyme well acted on aryl-β-d-xylosides, but showed no activity on alkyl-β-d-xylosides, and it was practically free from glucosidase activity. The Km and V_max values of this enzyme for xylobiose were calculated to be 2.86 × 10^?8 m and 34.5 μmoles/mg/min, respectively, and these values determined for phenyl-β-d-xyloside were 3.01 × 10^?8 m and 16.2 μmoles/mg/min, respectively.     

THE RESPONSE OF PER1 TO LIGHT IN THE SUPRACHIASMATIC NUCLEUS OF THE DIURNAL DEGU (OCTODON DEGUS)

Several studies suggest that the circadian systems of diurnal mammals respond differently to daytime light than those of nocturnal mammals. We hypothesized that the photosensitive “clock” gene Per1 would respond to light exposure during subjective day in the suprachiasmatic nucleus of the diurnal rodent, Octodon degus. Tissue was collected 1.5–2?h after a 30?min light pulse presented at five timepoints across the 24?h day and compared to controls maintained under conditions of constant darkness. Per1 mRNA was quantified using in situ hybridization. Results showed that the rhythmicity and photic responsiveness of Per1 in the degu resembles that of nocturnal animals. (Author correspondence: hagenaue@umich.edu)     

CHANGES IN POLYSOMES OF THE DEVELOPING RAT BRAIN

Abstract— Rat brain polysomes were prepared from a deoxycholate-treated postmito-chondrial supernatant in the presence of 2% bentonite and 1 mg/ml of yeast RNA to prevent partial degradation during preparation.

• 1 The polysomal preparations had an absorption maximum at 260 mμ and an absorption minimum at 235 mμ. The ratio of absorption maximum to minimum and the RNA to protein ratio were 1·58 and 1·06 respectively in 6-day-old rat brain polysomes. The sedimentation patterns showed six distinct peaks with sedimentation coefficients of 235S, 185S, 173S, 135S, 100S and 80S, indicating that these preparations have the characteristics of pure heavy polysomes.

• 2 The rate of [¹⁴C]phenylalanine incorporation into brain polysomal protein was maximal at approximately 10 days of age and decreased thereafter. A similar progressive reduction with increasing age was found in the stimulation of phenylalanine incorporation by the addition of 60 μg/tube of polyuridylic acid. However, the incorporation of phenylalanine into young rat brain polysomes was usually greater even with the addition of polyuridylic acid than in the older animals.

• 3 The comparative studies on sucrose density gradient centrifugation of polysomes between young and adult rat brains showed a considerable decrease of heavy polysomes in the older animals.

• 4 The effect of various factors on the stability of brain polysomes from both ages has been studied. The rates of RNA, protein and acid-soluble phosphorus release from polysomes of the adult rat brains were usually greater in the presence of high salt concentration, ethylenediaminetetra-acetic acid and urea than those from the corresponding preparations of younger animals. On the basis of evidence obtained from the above results it suggested that the adult brain polysomes were more unstable than those of younger animals.

• 5 The amount of polysomal RNA linearly increased up to the first 20 days after birth and then levelled off. The ratio of G + C/A + U of polysomal RNA was less in the young rat brains, falling to 1·30 as compared to 1·50 in older animals. The differences were statistically significant at less than a 1% level of confidence.

• 6 Polysomal preparations also contained RNase, phosphomonoesterase, phospho-diesterase and 5′-nucleotidase activities which cannot be washed off. The specific activities of these enzymes were generally higher in young rat brains than those in the adult.

     

Enzymatic Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

Some enzymatic properties of Malbranchea β-xylosidase were investigated. The β- xylosidase activity was inhibited by Hg²⁺, Zn²⁺, Cu²⁺, N-bromosuccinimide, p-chloromercuribenzoate and sodium laurylsulfate, while this activity was activated by Ca²⁺. The enzyme released xylose as the end product even from 10% xylobiose solution without forming any xylooligosaccharides. The enzyme well acted on aryl-β-d-xylosides, but showed no activity on alkyl-β-d-xylosides, and it was practically free from glucosidase activity. The Km and V_max values of this enzyme for xylobiose were calculated to be 2.86 × 10^?8 m and 34.5 μmoles/mg/min, respectively, and these values determined for phenyl-β-d-xyloside were 3.01 × 10^?8 m and 16.2 μmoles/mg/min, respectively.