Calcium pools,calcium entry,and cell growth

The Ca²⁺ pump and Ca²⁺ release functions of intracellular Ca²⁺ pools have been well characterized. However, the nature and identity of Ca²⁺ pools as well as the physiological implications of Ca²⁺levels within them, have remained elusive. Ca²⁺ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca²⁺ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca²⁺ pools, the levels of Ca²⁺ within Ca²⁺ pools, and how these intraluminal Ca²⁺ levels may be physiologically related to ER function. Experiments utilizingin situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluroescent Ca²⁺ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺ pools within cells, and large changes in the levels of Ca²⁺ within pools following InsP₃-mediated Ca²⁺ release. Such changes in Ca²⁺ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca²⁺-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca²⁺ changes with respect to cell growth. Emptying of pools using Ca²⁺ pump blockers can result in cells entering a stable quiescent G₀-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca²⁺ pools return, and cells reenter the cell cycle. During the Ca²⁺ pool-depleted growth-arrested state, cells express a Ca²⁺ influx channel that is distinct from the store-operated Ca²⁺ influx channels activated after short-term depletion of Ca²⁺ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca²⁺ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.     

Diel variability in nitrogenous nutrient uptake at photic and subphotic depths

Diel patterns in nitrogen uptake were investigated at threepositions along an offshore orientated transect in the SouthernBenguela Upwelling System. Both photic and sub-photic depthswere targeted in the ¹⁵N tracer experiments. An appreciableproportion of nitrogen uptake was found to occur at sub-photicdepths during both the day and the night. The importance ofsub-photic uptake decreased offshore but remained non-trivialat all locations. Although NH₄ dominated this uptake, NO₃ removalat depth did account for an important fraction of the totalwater column NO₃ uptake, particularly over the shelf at night.For example, sub-photic NO₃ uptake at night for the inshorestations averaged 68% of the total for the water column decliningto 3% offshore. The impact of sub-photic NO₄ uptake on new productionwill depend on whether the NO₃ is incorporated into primaryassimilation pathways or is transported to the sediments ininorganic form. Whereas sub-photic/photic uptake decreased offshore,night/day uptake ratios increased with distance from the coast,approaching unity at the offshore location. This effect wasmost obvious for the photic communities This finding supportsthe belief that dark nitrogen uptake assumes increased importanceas nutrients become less available. Photic zone f-ratios weresmaller at night (mean = 0.14) than during the day (mean = 0.28)indicating a greater importance of reduced nitrogen uptake duringthe dark hours This emphasis on NH₄ utilization at night canbe explained purely in terms of energy efficiency. Such die1phasing in f-ratios needs to be accounted for in scaling totalproduction to new proifudon.     

An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase (Xy1D) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls.

Diferulate esters strengthen and cross-link primary plant cell walls and help to defend the plant from invading microbes. Phenolics also limit the degradation of plant cell walls by saprophytic microbes and by anaerobic microorganisms in the rumen. We show that incubation of wheat and barley cell walls with ferulic acid esterase from Aspergillus niger (FAE-III) or Pseudomonas fluorescens (Xy1D), together with either xylanase I from Aspergillus niger, Trichoderma viride xylanase, or xylanase from Pseudomonas fluorescens (XylA), leads to release of the ferulate dimer 5-5' diFA [(E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid]. Direct saponification of the cell walls without enzyme treatment released the following five identifiable ferulate dimers (in order of abundance): (Z)-beta-(4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy)-4-hydroxy-3-methoxycinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl) -7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid, 5-5' diFA, (E,E)-4, 4'-dihydroxy-3, 5'-dimethoxy-beta, 3'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -6-methoxy-1, 2-dihydronaphthalene-2, 3-dicarboxylic acid. Incubation of the wheat or barley cell walls with xylanase, followed by saponification of the solubilized fraction, yielded 5-5'diFA and, in some cases, certain of the above dimers, depending on the xylanase used. These experiments demonstrate that FAE-III and XYLD specifically release only esters of 5-5'diFA from either xylanase-treated or insoluble fractions of cell walls, even though other esterified dimers were solubilized by preincubation with xylanase. It is also concluded that the esterified dimer content of the xylanase-solubilized fraction depends on the source of the xylanase.     

Diel Surface Temperature Range Scales with Lake Size

Ecological and biogeochemical processes in lakes are strongly dependent upon water temperature. Long-term surface warming of many lakes is unequivocal, but little is known about the comparative magnitude of temperature variation at diel timescales, due to a lack of appropriately resolved data. Here we quantify the pattern and magnitude of diel temperature variability of surface waters using high-frequency data from 100 lakes. We show that the near-surface diel temperature range can be substantial in summer relative to long-term change and, for lakes smaller than 3 km², increases sharply and predictably with decreasing lake area. Most small lakes included in this study experience average summer diel ranges in their near-surface temperatures of between 4 and 7°C. Large diel temperature fluctuations in the majority of lakes undoubtedly influence their structure, function and role in biogeochemical cycles, but the full implications remain largely unexplored.     

Nononcogenic deletion mutants of herpesvirus saimiri are defective for in vitro immortalization.

Herpesvirus saimiri L-DNA sequences between 0.0 and 4.0 map units (4.5 kilobase pairs) are required for oncogenicity; these sequences are not required for replication of the virus. To investigate the basis for the lack of oncogenicity of mutants with deletions in this region and to study the function of this region, we developed a reliable system for in vitro immortalization by herpesvirus saimiri. In contrast to peripheral blood lymphocytes from cotton-top tamarins (Saguinus oedipus) and owl monkeys (Aotus sp.), infection of peripheral blood lymphocytes from common marmosets (Callithrix jacchus) in vitro with herpesvirus saimiri consistently yielded continuously growing lymphoblastoid cell lines. Such cell lines were established using strains of herpesvirus saimiri from group A and group non-A, non-B; however, repeated attempts to immortalize common marmoset peripheral blood lymphocytes using strains from group B were not successful. Common marmoset cell lines immortalized by herpesvirus saimiri were T12+, T8+, T4-, and B1-, indicating that they were derived from suppressor/cytotoxic T lymphocytes. Cell lines could not be established using the nononcogenic mutants 11att and S4, both of which were derived from the group A strain 11 virus. Strain 11att has a spontaneous deletion and S4 has a constructed deletion in the 0.0 to 4.0 map unit region. Constructed strains which had these deleted sequences restored did immortalize common marmoset peripheral blood lymphocytes. Thus, the nononcogenic deletion mutants are defective for immortalization. This system should facilitate attempts to define the sequences responsible for immortalization and to determine their function.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Control of invertase synthesis in sugar cane. Loci of auxin and glucose effects

In tissue slices from rapidly expanding internodes of sugar cane the vacuolar invertase level is a function of the balance between synthesis and destruction. The enzyme is destroyed in the tissue at an approximately constant rate with a half time of 2 hours. Invertase synthesis is regulated by both auxin and glucose. From studies with inhibitors of protein and RNA synthesis we conclude that auxin alters the rate of synthesis and glucose increases the rate of destruction of messenger RNA required for the production of invertase.     

The solubilization of a glucuronyltransferase involved in pea (Pisum sativum var. Alaska) glucuronoxylan synthesis.

A glucuronyltransferase involved in glucuronoxylan biosynthesis was obtained from the epicotyls of 1-week-old etiolated pea (Pisum sativum var. Alaska) seedlings and was solubilized in Triton X-100, a non-ionic detergent. The enzyme was inactivated by SDS and inhibited by Derriphat 160 and cholic acid. The enzyme was active in the presence of NN-dimethyldodecylanium-N-oxide, but was not solubilized by it. The stimulatory effect of UDP-D-xylose on the particulate and solubilized enzymes was the same, but the optimum Mn2+ concentration was lower for the solubilized enzyme, and the product formed by the solubilized enzyme has altered structure and solubility properties. Gel filtration of the solubilized enzyme on Sepharose CL-6B permitted partial separation of the stimulatory effect of UDP-D-xylose from the activity in the absence of UDP-D-xylose. The solubilized enzyme was more stable than the particulate enzyme and could be stored for 2 weeks at -20 degrees C without loss of activity.     

α‐secretase mediated conversion of the amyloid precursor protein derived membrane stub C99 to C83 limits Aβ generation

The Swedish mutation within the amyloid precursor protein (APP) causes early‐onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While β‐secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild‐type APP occurs mainly in endocytic compartments. However, we show that liberation of Aβ from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall β‐secretase cleaved soluble APP released from APP_swe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased Aβ secretion. We point out that α‐secretase activity‐mediated conversion of C99 to C83 is the main cause of this Aβ reduction. Furthermore, we demonstrate that α‐secretase cleavage of C99 even contributes to the reduction of Aβ secretion of internalization deficient wild‐type APP. Therefore, inhibition of α‐secretase cleavage increased Aβ secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells.