Limnology of the Crater Lake Cuicocha,Ecuador, a Cold Water Tropical Lake

Cuicocha (3380 m a.s.l.) is a young, a few hundred years old volcanic lake in the western cordilleras of the Ecuadorian Andes with some post‐volcanic activities, such as emission of volcanic gases and input of hydrothermal water. Water chemistry is influenced by the emission of CO₂ and weathering of the young andesitic rocks in the water shed. A calcium cycle exists in the lake with intensive biological Ca precipitation at the flanks and formation of travertine crusts, while in the hypolimnion dissolution of Ca carbonate occurs. The crater lake is oligotrophic, biodiversity is low; the littoral flora and fauna is more important than the pelagic species. In the littoral zone, a small Totora zone occurs, followed by submerged macrophytes down to 35 m water depth. Phyto‐ and zooplankton occur down into the hypolimnion. Phytoplankton is strongly influenced by down‐welling of water (atelomixis) and by coprecipitation with detritial flocs (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking

A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in lysosomes. The Hrs-STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs-STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.     

Mutational analysis of putative helix-helix interacting GxxxG-motifs and tryptophan residues in the two-peptide bacteriocin lactococcin G

The membrane-permeabilizing two-peptide bacteriocin lactococcin G consists of two different peptides, LcnG-alpha and LcnG-beta. The bacteriocin contains several tryptophan and tyrosine residues and three putative helix-helix interacting GxxxG-motifs, G 7xxxG 11 and G 18xxxG 22 in LcnG-alpha and G 18xxxG 22 in LcnG-beta. The tryptophan and tyrosine residues and residues in the GxxxG-motifs were altered by site-directed mutagenesis to analyze the structure and membrane-orientation of lactococcin G. Substituting the glycine residues at position 7 or 11 in the G 7xxxG 11-motif in LcnG-alpha with large hydrophobic or hydrophilic residues was highly detrimental, whereas small residues were tolerated. Qualitatively similar results were obtained for the G 18xxxG 22-motif in LcnG-beta. In contrast, replacement of the glycine residues in the middle of these two motifs with large hydrophilic residues was tolerated. All mutations in the G 18xxxG 22-motif in LcnG-alpha were relatively well-tolerated, indicating that this motif is not involved in helix-helix interactions. The four aromatic residues in the N-terminal part of LcnG-beta could individually be replaced by other aromatic residues, a hydrophilic positive residue, and a hydrophobic residue without a marked reduced activity, indicating that this region is structurally flexible and not embedded in a strictly hydrophobic or hydrophilic environment. The results are in accordance with a structural model where the G 7xxxG 11-motif in LcnG-alpha and the G 18xxxG 22-motif in LcnG-beta interact and allow the two peptides to form a parallel transmembrane helix-helix structure, with the tryptophan-rich N-terminal part of LcnG-beta positioned in the outer membrane interface and the cationic C-terminal end of LcnG-alpha inside the cell.     

Chk2 Activation Dependence on Nbs1 after DNA Damage

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.     

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h⁻¹ g (dry weight) of cells⁻¹ (0.24 to 0.30 g h⁻¹ g [dry weight] of cells⁻¹) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h⁻¹. The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

Remote prey detection in Oithona similis: hydromechanical versus chemical cues

We quantified prey encounter rates and prey reaction distancesin the ambush-feeding cyclopoid copepod Oithona similis by videorecording freely swimming copepods at different concentrationsof prey, the dinoflagellate Gymnodinium dominans. Prey encounterrate increased with prey concentration, and a maximal clearancerate of 0.42 ± 0.10 ml h^–1 was estimated. The averagedistance (from the antennules) at which O.similis reacts toprey is 0.014 ± 0.007 cm. A simple prey encounter modelwas used to combine observed predator and prey velocities andprey reaction distance, and yielded a clearance rate similarto that estimated directly from prey encounter rates. The observedprey reaction distance was consistent with that estimated froma published model of hydromechanical prey perception. The possibilityof remote chemodetection was examined by modeling the distributionof solutes leaking out of a swimming cell. The cell leaves along slender chemical trail in its wake. However, since theambush-feeding O.similis is essentially stationary when perceivingprey, it is the width rather than the length of the trail thatmatters. Owing to advection, the chemical signal vanishes almostinstantaneously off the sides of the swimming flagellate, andsolute concentrations are below any likely detection thresholdwithin 40–50 µm from the flagellate. Our observationsare thus inconsistent with remote chemodetection in O.similis.The considerations are generalized, and it is concluded thatambush-feeding copepods, unlike cruisers and suspension feeders,cannot utilize chemical signals for the detection of individualprey, but rely on either hydromechanical detection or directinterception of prey.     

A new side to ubiquitin

Mono-ubiquitination is a common mechanism of protein regulation, and more than ten ubiquitin-interacting domains that recognize the hydrophobic region centered on Ile44 of ubiquitin have been characterized. Two recent reports describe the crystal structure of the Rab5 guanine-nucleotide-exchange factor Rabex-5 and show that it contains two novel ubiquitin-binding domains. One of these is an A20 zinc finger that binds to a polar interaction interface of ubiquitin centered on Asp58. The discovery of an alternative interaction face of ubiquitin opens new avenues for understanding how this small protein regulates protein function.     

Energy supply of the okapi in captivity: fermentation characteristics of feedstuffs

A variety of feeds are used in the nutrition of browsing ruminants. During digestion trials on okapis, feedstuffs of different facilities were sampled and the Hohenheim gas test was used as in vitro fermentation method to quantify their fermentative behavior. Forty‐six feeds were analyzed (7, fruit and vegetable; 11, energy concentrates and pelleted compounds; 13, forage; 9, browse leaf; 6, small and large twig samples). Gas production of these samples was recorded after 2, 4, 6, 8, 10, 12, and 24 hr of fermentation. Browse leaf samples were additionally analyzed with a tannin‐binding agent (polyethylene‐glycol) to assess limiting effects of condensed tannins. Metabolizable energy (ME) was estimated from 24 hr gas production according to standard regressions. Vegetables and particularly fruits were found to yield very high gas productions during the first 2 hr of fermentation, whereas unmolassed beet pulp was found to have a more even distribution of gas production/energy release over total fermentation time. Feeds like rolled oats or bread were evaluated to yield very high energy contents of >14 MJ ME/kg dry matter (DM). Alfalfa (Medicago sativa) hay had a comparable fermentation pattern to fresh browse samples, characterized by a high fermentation rate. In conclusion, energy‐rich constituents for captive ruminant diets should not include larger amounts of vegetables and especially fruits, due to their very fast fermentation during the initial phase of fermentation and the connected risk of rumen acidosis. Energy‐concentrates like beet pulp (unmolassed) showed moderate fermentation characteristics and energy content and are well suited as a component of zoo ruminant diets. Energy‐concentrates with very high energy densities (>13 MJ ME/kg DM) like bread or rolled oats are not suitable for a diet that is intended to promote long feeding times. Various aspects are involved in the decision for appropriate forage for browsing ruminants; based on fermentation pattern, alfalfa hay seems to be a reasonable substitute for browse leaves. Zoo Biol 0:1–16, 2006. © 2006 Wiley‐Liss, Inc.