Embryonal neural tumours and cell death

Medulloblastoma and neuroblastoma are malignant embryonal childhood tumours of the central and peripheral nervous systems, respectively, which often show poor clinical prognosis due to resistance to current chemotherapy. Both these tumours have deficient apoptotic machineries adopted from their respective progenitor cells. This review focuses on the specific background for tumour development, and highlights biological pathways that present potential targets for novel therapeutic approaches.     

MADS about MOSS

Classic MIKC-type MADS-box genes (MIKC^c) play diverse and crucial roles in angiosperm development, the most studied and best understood of which is the specification of floral organ identities. To shed light on how the flower evolved, phylogenetic and functional analyses of genes involved in its ontogeny, such as the MIKC^c genes, must be undertaken in as broad a selection as possible of plants with disparate ancestries. Since little is known about the functions of these genes in non-seed plants, we investigated the developmental roles of a subset of the MIKC^c genes present in the moss, Physcomitrella patens, which is positioned informatively near the base of the land plant evolutionary tree. We observed that transgenic lines possessing an antisense copy of a MIKC^c gene characteristically displayed knocked-down expression of the corresponding native MIKC^c gene as well as multiple diverse phenotypic alterations to the haploid gametophytic and diploid sporophytic generations of the life cycle.¹ In this addendum, we re-examine our findings in the light of recent pertinent literature and provide additional data concerning the effects of simultaneously knocking out multiple MIKC^c genes in this moss.Key words: Physcomitrella, moss, MADS-box gene functions, gene knock-down, gene knockout, gene expression, evo-devoThe moss, Physcomitrella patens, is the only non-seed plant that is amenable to an investigation of MADS-box gene function comparable to that achieved in angiosperms. P. patens possesses six MIKC^c genes which cluster into two distinct phylogenetic clades.² We recently reported a functional genetic analysis of the three genes (PPM1, PPM2 and PpMADS1) within the PPM2-like clade as an initial contribution towards gaining an understanding of the role(s) of MIKC^c genes in this moss.¹By fusing the respective MIKC^c promoters to a GUS reporter gene, we found that both PPM1 and PpMADS1 exhibited fairly ubiquitous expression patterns in both gametophytic and sporophytic tissues. The levels of PPM1 expression were generally higher than those of PpMADS1, and PpMADS1 was not expressed in antheridia, suggesting subtle differences in the functions of these genes. The observed patterns of widespread expression resemble those characterising the majority of vascular, non-seed plant MIKC^c genes^3–7 and accord with RT-PCR results of Quodt and coworkers.² Our in situ GUS expression and RT-PCR results¹ showed that PPM2 was not expressed or was expressed at levels too low to be detected by these methods. Conversely, the original isolation of PPM2 cDNA⁸ and data from more recent expression studies² indicated that PPM2 is expressed (albeit inconsistently and weakly) ubiquitously with elevated levels of expression sometimes observed in gametangia, sporophytic feet and basal portions of sporophytic setae.² The contradictory expression data for PPM2 may derive from differences between the PPM2-reporter gene constructs used by the respective research groups^1,2 or perhaps from variations in moss culture conditions.We also employed an antisense approach designed to knock down expression of PPM1, and perhaps closely related MIKC^c genes, in order to discern MADS-box gene function in P. patens. Knocked-down strains displayed a complex mutant phenotype comprising delayed gametangia formation and sporophyte production, diminished sporophyte yields, and morphological abnormalities in both leaves and sporophytes, findings that are generally consistent with the ubiquitous expression pattern of PPM1¹ and PPM2''s expression as described by Quodt et al.²The phenotypes of strains with single gene knockouts of PPM1, PPM2 or PpMADS1 appeared to be perfectly normal, not displaying any of the phenotypic alterations observed in PPM1 gene knock-down mutants. While it is possible that subtle, transient or conditional phenotypic changes went unnoticed, it seems more probable that genetic redundancy is responsible for these results since the PPM2-like genes exhibit a very high level of sequence similarity. In an effort to circumvent the problem of functional redundancy, we generated all double knockout combinations for PPM1, PPM2 and PpMADS1. However, the double mutants were also phenotypically unchanged. Finally we attempted to produce triple mutants by co-transforming single PPM2 knockout lines with PPM1 and PpMADS1 linear knockout constructs. Of the 31 stable transformants from two transformation experiments, 55% were shown to be double mutants in which the original PPM2 knockout was accompanied by a second gene knockout in either PPM1 or PpMADS1. However, no triple knockouts were obtained. Given the knockout frequencies generally observed in batch transformation experiments in our laboratory and those of others,⁹ between two and five of the transformants had been expected to be triple mutants. These preliminary data, albeit involving a relatively small sample of transformants, suggest that PPM1, PPM2 and PpMADS1 triple knockouts may be lethal.We have related compelling evidence that functionally redundant PPM2-like MIKC^c genes are involved in several aspects of the moss developmental program. It has been argued that broad expression patterns like theirs represent the ancestral state of MADS-box genes in land plants, and that the sporophytic- and organ-specific expression patterns that characterise many MIKC^c genes in extant spermatophytes, including those that specify floral organ identity, correspond to a derived condition that evolved in the spermatophyte lineage following its separation from lineages that led to bryophytes and ferns and fern allies.¹⁰ Nevertheless, it is the apparent participation of PPM2-like genes in the formation of gametangia (the differentiation of reproductive organs from non-reproductive tissues at the gametophore apex) that is particularly interesting and assumes a special significance because of its analogy to the proposed role for ancestors of seed plant C-function MADS-box genes (identifying those regions of the vegetative SAM that will become reproductive organs).11 Furthermore, expression studies of MIKC^c genes in two charophycean algae, the presumed progenitors of all terrestrial plants,^12–14 suggest that they too are involved in haploid reproductive cell differentiation.¹⁵ While these functional similarities do not infer orthology and may be coincidental, we should not discount yet the admittedly controversial hypothesis that some MIKC^c genes in non-seed plants, for example PPM2-like genes of Physcomitrella, are homologous to spermatophyte class C genes and that the ancient role proposed for ancestral class C genes¹¹ has been conserved, in some form, in all major terrestrial plant taxa.     

Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

The 26 S proteasome is a large proteolytic machine, which degrades most intracellular proteins. We found that thioredoxin, Txnl1/TRP32, binds to Rpn11, a subunit of the regulatory complex of the human 26 S proteasome. Txnl1 is abundant, metabolically stable, and widely expressed and is present in the cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential of about-250 mV. Mutant Txnl1 with one active site cysteine replaced by serine formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26 S proteasome. eEF1A1 is therefore a likely physiological substrate. In response to knockdown of Txnl1, ubiquitin-protein conjugates were moderately stabilized. Hence, Txnl1 is the first example of a direct connection between protein reduction and proteolysis, two major intracellular protein quality control mechanisms.Degradation of proteins in eukaryotic cells plays a pivotal role in the regulation of several important processes, including cell division, antigen presentation, and signal transduction (1). Most intracellular proteins are degraded by the 26 S proteasome, a 2.5-MDa protease complex composed of more than 30 different subunits (2).To become degraded, proteins are typically first conjugated to a chain of ubiquitin moieties. This reaction is catalyzed by ubiquitin ligases. The ubiquitin chains lend the proteins affinity for the 26 S proteasome (3). For efficient degradation, certain ubiquitylated proteins are shuttled to the 26 S proteasome by substrate recruiting factors, such as Rad23, Dsk2, and eEF1A (4, 5).The 26 S proteasome is composed of two stable subcomplexes, the proteolytically active 20 S core and 19 S regulatory complexes, which bind to one or both ends of the cylindrical 20 S core particle (6). The regulatory complexes first recognize the ubiquitylated substrates (3), before the substrates are deubiquitylated (7, 8), unfolded (9, 10), and translocated into the 20 S particle for degradation.Although the 26 S proteasome has been known for more than 20 years (11), novel subunits and cofactors have been described recently (12, 13). Here we report another novel proteasome-associated protein, Txnl1 (thioredoxin-like protein 1), that associates directly with the proteasome subunit Rpn11. Txnl1 exhibits thioredoxin activity and targets eEF1A1 in vivo. Previous reports have shown that eEF1A1 transfers misfolded nascent proteins from the ribosome to the 26 S proteasome for degradation (5, 14, 15). Accordingly, ubiquitin-protein conjugates were stabilized upon knockdown of Txnl1 expression. Txnl1 therefore directly links protein reduction and proteolysis, two major intracellular protein quality control mechanisms.     

Influence of sea temperature and initial marine feeding on survival of Atlantic salmon Salmo salar post-smolts from the Rivers Orkla and Hals, Norway

The abundance of returning adult Atlantic salmon Salmo salar, in the River Orkla in mid‐norway (1 sea‐winter, SW, fish) and River Hals in north Norway (1–3 SW fish), was tested against the early marine feeding and the seawater temperature experienced by their corresponding year classes of post‐smolts immediately after entry into the Trondheimsfjord (Orkla smolts, 22 years of data) and Altafjord (Hals smolts, 17 years of data). In both river–fjord systems, there was a significant positive correlation between the abundance of returning S. salar and the mean seawater temperature at the time of smolts descending to the sea. The number of 1SW fish reported caught in River Orkla was positively correlated to the proportion of fish larvae in the post‐smolt stomachs in Trondheimsfjord. The abundance of returning S.salar was, however, neither correlated to forage ratio (R_F) nor other prey groups in post‐smolt stomachs in the two fjord systems. In the Altafjord, the post‐smolts fed mainly on pelagic fish larva (70–98%) and had a stable R_F (0·009–0·023) over the 6 years analysed. In the Trondheimsfjord, however, there was a higher variation in R_F (0·003–0·036), and pelagic fish larvae were dominant prey in only two (50 and 91%) of the 8 years analysed. These 2 years also showed the highest return rates of S. salar in River Orkla. These results demonstrate that the thermal conditions experienced by post‐smolts during their early sea migration may be crucial for the subsequent return rate of adults after 1–3 years at sea. Pelagic marine fish larvae seem to be the preferred initial prey for S. salar post‐smolts. As the annual variation in abundance of fish larvae is related to seawater temperature, it is proposed that seawater temperature at sea entry and the subsequent abundance of returning adult S. salar may be indirectly linked through variation in annual availability of pelagic fish larvae or other suitable food items in the early post‐smolt phase.     

Sexual Isolation in Acinetobacter baylyi Is Locus-Specific and Varies 10,000-Fold Over the Genome

Naturally transformable bacteria acquire chromosomal DNA from related species at lower frequencies than from cognate DNA sources. To determine how genome location affects heterogamic transformation in bacteria, we inserted an nptI marker into random chromosome locations in 19 different strains of the Acinetobacter genus (>24% divergent at the mutS/trpE loci). DNA from a total of 95 nptI-tagged isolates was used to transform the recipient Acinetobacter baylyi strain ADP1. A total of >1300 transformation assays revealed that at least one nptI-tagged isolate for each of the strains/species tested resulted in detectable integration of the nptI marker into the ADP1 genome. Transformation frequencies varied up to ∼10,000-fold among independent nptI insertions within a strain. The location and local sequence divergence of the nptI flanking regions were determined in the transformants. Heterogamic transformation depended on RecA and was hampered by DNA mismatch repair. Our studies suggest that single-locus-based studies, and inference of transfer frequencies from general estimates of genomic sequence divergence, is insufficient to predict the recombination potential of chromosomal DNA fragments between more divergent genomes. Interspecies differences in overall gene content, and conflicts in local gene organization and synteny are likely important determinants of the genomewide variation in recombination rates between bacterial species.HORIZONTAL gene transfer (HGT) contributes to bacterial evolution by providing access to DNA evolved and retained in separate species or strains (Cohan 1994a,b; Bergstrom et al. 2000; Ochman et al. 2000; Feil et al. 2001; Koonin 2003; Lawrence and Hendrickson 2003; Fraser et al. 2007). Multilocus sequence typing (MLST) has provided strong evidence for frequent transfer and recombination of chromosomal DNA between related bacterial strains within the same species (Maiden et al. 1998; Enright et al. 2002). HGT occurring by natural transformation allows bacteria to exploit the presence of nucleic acids in their environment for the purposes of nutrition, DNA repair, reacquisition of lost genes, and/or acquisition of novel genetic diversity (Redfield 1993; Mehr and Seifert 1998; Dubnau 1999; Claverys et al. 2000; Szöllösi et al. 2006; Johnsen et al. 2009). It can be inferred from observations of the presence of extracellular DNA in most environments that bacteria are constantly exposed to DNA from a variety of sources, without such exposure necessarily producing observable changes in the genetic compositions of bacterial populations over evolutionary time (Thomas and Nielsen 2005; Nielsen et al. 2007a,b).The absence of sequence similarity between the donor DNA and the DNA of the recipient bacterium is the strongest barrier to the horizontal acquisition of chromosomal genes in bacteria (Matic et al. 1996; Vulic et al. 1997; Majewski 2001; Townsend et al. 2003) as illegitimate recombination occurs only at extremely low frequencies in bacteria (Hülter and Wackernagel 2008a). Single-locus transfer models have been extensively applied and have demonstrated a log-linear decrease in recombination frequencies with increasing sequence divergence for Bacillus subtilis (Roberts and Cohan, 1993; Zawadzki et al. 1995), Acinetobacter baylyi (Young and Ornston 2001), Escherichia coli (Shen and Huang 1986; Vulic et al. 1997), and Streptococcus pneumoniae (Majewski et al. 2000). For instance, heterogamic transformation between nonmutator isolates at the rpoB locus of B. mojavensis is undetectable at sequence divergences >16.7% (Zawadzki et al. 1995) and between S. pneumoniae isolates with sequence divergences >18% (Majewski et al. 2000). In A. baylyi, the nonmutator sequence divergence limit for detectable transformation at the pcaH locus of strain ADP1 was found to be 20% (Young and Ornston 2001), and up to 24% overall divergence yielded transformants at 16S rRNA loci in strain DSM587 (Strätz et al. 1996).Several recent studies also show that short stretches (<200 bp) of DNA sequence identity can facilitate additive or substitutive integration of longer stretches (>1000 bp) of heterologous DNA in bacteria (Prudhomme et al. 1991, 2002; de Vries and Wackernagel 2002; Hülter and Wackernagel 2008a). Thus, the uptake of DNA in bacteria can facilitate larger substitutions within gene sequences and the integration of additional DNA material on the basis of recombination initiated in flanking DNA stretches (either at one or both ends) with high sequence similarity (Nielsen et al. 2000). On the other hand, segments of heterologous DNA interrupting the synteny of homologous DNA have also been shown to be a barrier in intraspecies transformation in S. pneumoniae (Pasta and Sicard 1996, 1999).The various studies of the interspecies transfer potential of single genes demonstrate that the immediate local sequence divergence of the transferred locus is of high importance in determining recombination frequencies in hosts up to 20% divergent (at the housekeeping gene level). However, it can be hypothesized that the broader structural, organizational, and biochemical properties of the genome region surrounding a particular locus will determine its transfer potential to more divergent host species (Cohan 2001; Lawrence 2002). The interspecies transfer potential of various genome regions/loci between more diverged species (>20% at the housekeeping gene level) may therefore differ substantially from a log-linear model (determined experimentally for more closely related species) as local gene organization becomes less conserved with evolutionary time. The barriers to gene exchange between divergent bacterial species is likely a combination of inefficient recombination due to both mismatched base pairs (the main determinator in the log-linear model) and conflicting gene order and organization across the local recombining DNA regions. In addition, selective barriers due to negative effects on host fitness of the transferred DNA regions may become increasingly important for the removal of recombination events from the bacterial population. Recent bioinformatics-based genome analysis of E. coli and Salmonella genomes suggests various parts of the bacterial genome may have different suceptibilities to undergo evolutionarily successful recombination leading to temporal fragmentation of speciation (Lawrence 2002; Retchless and Lawrence 2007). Nevertheless, few studies have experimentally tested the effect of variable species and chromosome locations of genes on their transfer potential between bacteria (Ravin and Chen 1967; Ravin and Chakrabarti 1975; Siddiqui and Goldberg 1975; Cohan et al. 1991; Huang et al. 1991; Fall et al. 2007).Here, we determine to what extent genome location contributes to sexual isolation between the recipient A. baylyi strain ADP1 and 19 sequence divergent (24–27% divergent at the mutS/trpE loci) donor Acinetobacter strains and species (carrying a selectable nptI gene in a total of 95 random genome locations).     

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following ¹³C-labeling patterns of proteinogenic amino acids after growth on [¹³C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.d-Xylose, a constituent of the polymer xylan, is the major component of the hemicellulose plant cell wall material and thus one of the most abundant carbohydrates in nature. The utilization of d-xylose by microorganisms has been described in detail in bacteria and fungi, for which two different catabolic pathways have been reported. In many bacteria, such as Escherichia coli, Bacillus, and Lactobacillus species, xylose is converted by the activities of xylose isomerase and xylulose kinase to xylulose 5-phosphate as an intermediate, which is further degraded mainly by the pentose phosphate cycle or phosphoketolase pathway. Most fungi convert xylose to xylulose 5-phosphate via xylose reductase, xylitol dehydrogenase, and xylulose kinase. Xylulose 5-phosphate is also an intermediate of the most common l-arabinose degradation pathway in bacteria, e.g. of E. coli, via activities of isomerase, kinase, and epimerase (1).Recently, by genetic evidence, a third pathway of xylose degradation was proposed for the bacterium Caulobacter crescentus, in analogy to an alternative catabolic pathway of l-arabinose, reported for some bacteria, including species of Azospirillum, Pseudomonas, Rhizobium, Burkholderia, and Herbasprillum (2, 3). In these organisms l-arabinose is oxidatively degraded to α-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of l-arabinose dehydrogenase, l-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-l-arabinoate and α-ketoglutarate semialdehyde; the latter compound is further oxidized to α-ketoglutarate via NADP⁺-specific α-ketoglutarate semialdehyde dehydrogenase (KGSADH).² In a few Pseudomonas and Rhizobium species, a variant of this l-arabinose pathway was described involving aldolase cleavage of the intermediate 2-keto-3-deoxy-l-arabinoate to pyruvate and glycolaldehyde, rather than its dehydration and oxidation to α-ketoglutarate (4). Because of the presence of some analogous enzyme activities in xylose-grown cells of Azosprillum and Rhizobium, the oxidative pathway and its variant was also proposed as a catabolic pathway for d-xylose. Recent genetic analysis of Caulobacter crecentus indicates the presence of an oxidative pathway for d-xylose degradation to α-ketoglutarate. All genes encoding xylose dehydrogenase and putative lactonase, xylonate dehydratase, 2-keto-3-deoxylonate dehydratase, and KGSADH were found to be located on a xylose-inducible operon (5). With exception of xylose dehydrogenase, which has been partially characterized, the other postulated enzymes of the pathway have not been biochemically analyzed.The pathway of d-xylose degradation in the domain of archaea has not been studied so far. First analyses with the halophilic archaeon Haloarcula marismortui indicate that the initial step of d-xylose degradation involves a xylose-inducible xylose dehydrogenase (6) suggesting an oxidative pathway of xylose degradation to α-ketoglutarate, or to pyruvate and glycolaldehyde, in analogy to the proposed oxidative bacterial pentose degradation pathways. Recently, a detailed study of d-arabinose catabolism in the thermoacidophilic crenarchaeon Sulfolobus solfataricus was reported. d-Arabinose was found to be oxidized to α-ketoglutarate involving d-arabinose dehydrogenase, d-arabinoate dehydratase, 2-keto-3-deoxy-d-arabinoate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase (3).In this study, we present a comprehensive analysis of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. This halophilic archaeon was chosen because it exerts several suitable properties for the analyses. For example, it can be cultivated on synthetic media with sugars, e.g. xylose, an advantage for in vivo labeling studies in growing cultures. Furthermore, a shotgun DNA microarray of H. volcanii is available (7) allowing the identification of xylose-inducible genes, and H. volcanii is one of the few archaea for which an efficient protocol was recently described to generate in-frame deletion mutants.Accordingly, the d-xylose degradation pathway was elucidated following in vivo labeling experiments with [¹³C]xylose, DNA microarray analyses, and the characterization of enzymes involved and their encoding genes. The functional involvement of genes and enzymes was proven by constructing corresponding in-frame deletion mutants and their analysis by selective growth experiments on xylose versus glucose. The data show that d-xylose was exclusively degraded to α-ketoglutarate involving xylose dehydrogenase, a novel xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase.     

Quantitative characteristics of Atlantic halibut, Hippoglossus hippoglossus L., semen throughout the reproductive season

The overall objective of the study was to investigate changes in quantitative parameters of Atlantic halibut (Hippoglossus hippoglossus L.) semen throughout the reproductive season in order to systematize the knowledge about biology of Atlantic halibut spermatozoa. Semen samples were collected from February to May from broodstock males kept under either a natural or 3-month advanced photoperiod regime. Spermatozoa concentration, semen pH and osmolality, as well as spermatozoa motility parameters were investigated. The use of catheterization of sperm was examined. Also, fertilization tests were performed. We found that spermatozoa concentration increases in a linear-like mode towards the end of the spawning season, which correlated with a decrease in a number of spermatozoa motility parameters, including actual percentage of motile spermatozoa (MOT), curvilinear velocity (VCL) and straight-line velocity (VSL) of spermatozoa. A breakpoint in MOT occurred when spermatozoa reached a concentration in the range of 17-20 x 10(9) spermatozoa/mL. The fertilization ability of sperm from males kept under natural photoperiod decreased in April. Survival of embryos at 80 degrees days produced by fertilizing eggs of single female with sperm from natural photoperiod males was 88, 76 and 41% on April 09 and 17, and May 01, respectively, whereas using sperm from 3-month delayed photoperiod males for fertilizing eggs from the same female on April 27 resulted in 80% of surviving embryos, not differing significantly from the data from April 09. Physical decomposition of spermatozoa was observed towards the end of the season and it was related to an increase in the whole semen osmolality. Catheterization of semen did not improve spermatozoa motility parameters, however, it reduced the variation in recorded values, especially in the case of pH, caused by contamination with feces or urine. Post-seasonal decrease in spermatozoa concentration was likely related to intensive ageing processes. Based on the present study and available data by other researchers, a model of changes of quantitative parameters in Atlantic halibut semen throughout the reproductive season is proposed.     

Secreted β-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 3.2.1.23) at temperatures below 25°C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term stability at temperatures above 25°C.     

Isolation and characterization of human apolipoprotein M-containing lipoproteins

Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; approximately 5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDL(apoM+)) contained significantly more free cholesterol than HDL lacking apoM (HDL(apoM-)) (5.9 +/- 0.7% vs. 3.2 +/- 0.5%; P < 0.005) and was heterogeneous in size with both small and large particles. HDL(apoM+) inhibited Cu(2+)-induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDL(apoM-). In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM.