Enriching marine macroalgae with eicosatetraenoic (arachidonic) and eicosapentaenoic acids by chilling

Summary Twelve macroalgae belonging to the Chlorophyta, Phaeophyta and Rhodophyta were collected from the Arabian Gulf. Field samples and samples that were first incubated at 5° C and 24° C in the light for 1 week were analysed for lipids and fatty acids. The lipid contents varied according to the macroalga and, within the Chlorophyta and Phaeophyta, some representatives accumulated more lipids at 5° C and others at 24° C. All samples of algae had similar lipid composition with only quantitative differences. The temperature did not have a common effect on the lipid composition of representative algae, although changes in the relative concentration of specific classes were recorded. The Phaeophyta and Rhodophyta were as a rule richer than the Chlorophyta in eicosatetranoic (20:4) and eicosapentaenoic (20:5) but poorer in linolenic (18:3) acids. In most of the algae, incubation at 5° C was associated with lowering the proportion of palmitic acid (16:0) in the total lipids, and, but only in the Phaeophyta and Rhodophyta, increasing the concentration of 20:4 and 20:5. These polyunsaturated fatty acids occurred in high levels in monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG) of the Phaeophyta and Rhodophyta but not the Chlorophyta, the MGDG and DGDG of which were rich in 18:3 and hexadecatrienoic acid (16:3). Offprint requests to: R. H. Al-Hasan     

In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme

The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined.     

Validated high-performance liquid chromatographic method utilizing solid-phase extraction for the simultaneous determination of naringenin and hesperetin in human plasma

Naringenin and hesperetin, the aglycones of the flavanone glucosides naringin and hesperidin occur naturally in citrus fruits. They exert a variety of pharmacological effects such as antioxidant, blood lipid-lowering, anticarcinogenic and inhibit selected cytochrome P-450 enzymes resulting in drug interactions. A specific, sensitive, precise, and accurate solid-phase extraction high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of naringenin and hesperetin in human plasma was developed and validated. After addition of 7-ethoxycoumarin as internal standard, plasma samples were incubated with beta-glucuronidase/sulphatase, and the analytes were isolated from plasma by solid-phase extraction using C(18) cartridges and separated on a C(8) reversed phase column with methanol/water/acetic acid (40:58:2, v/v/v) as the eluent at 45 degrees C. The method was linear in the 10-300 ng/ml concentration range for both naringenin and hesperetin (r>0.999). Recovery for naringenin, hesperetin and internal standard was greater than 76.7%. Intra- and inter-day precision for naringenin ranged from 1.4 to 4.2% and from 1.9 to 5.2%, respectively, and for hesperetin ranged from 1.3 to 4.1% and from 1.7 to 5.1%, respectively. Accuracy was better than 91.5 and 91.3% for naringenin and hesperetin, respectively.     

Butyrylcholinesterase Predicts Cardiac Mortality in Young Patients with Acute Coronary Syndrome

Background

The incidence of acute coronary syndrome (ACS) in young people (≤65 years) is continuously rising. While prognostic factors in ACS are well-investigated less attention has been paid to their age-dependent prognostic value and their particular relevance in younger patients. The aim of our study was to assess the age-dependent prognostic impact of butyrylcholinesterase (BChE).

Methods

Retrospective cohort study including 624 patients with ACS. Patients were stratified by age into equal groups (n = 208) corresponding to “young patients” (45–64 years), "middle-aged patients” (65–84 years) and “old patients” (85–100 years). Cox regression hazard analysis was used to assess the influence of BChE on survival.

Results

After a mean follow-up time of 4.0 (interquartile range [IQR] 2.0–6.4) years, 154 patients (24.7%) died due to a cardiac cause. In the overall cohort, BChE was indirectly associated with cardiac mortality-free survival (adjusted hazard ratio (HR): 0.70 (95% confidence interval [CI] 0.53–0.93, p = 0.01). The primary-analysis of BChE by age strata showed the strongest effect in the age group 45–64 years with an adjusted HR per 1-SD of 0.28 (95% CI 0.12–0.64, p = 0.003), a weaker association with mortality in middle aged (65–84 years: adjusted HR per 1-SD 0.66 [95% CI: 0.41–1.06], p = 0.087), and no association in older patients (85–100 years: adjusted HR per 1-SD 0.89 [95% CI: 0.58–1.38], p = 0.613).

Conclusion

BChE is a strong predictor for cardiac mortality specifically in younger patients with ACS aged between 45 and 64 years. No significant association of BChE with cardiac-mortality was detected in other age classes.     

Neurodevelopmental consequences of sub-clinical carbon monoxide exposure in newborn mice

Carbon monoxide (CO) exposure at high concentrations results in overt neurotoxicity. Exposure to low CO concentrations occurs commonly yet is usually sub-clinical. Infants are uniquely vulnerable to a variety of toxins, however, the effects of postnatal sub-clinical CO exposure on the developing brain are unknown. Apoptosis occurs normally within the brain during development and is critical for synaptogenesis. Here we demonstrate that brief, postnatal sub-clinical CO exposure inhibits developmental neuroapoptosis resulting in impaired learning, memory, and social behavior. Three hour exposure to 5 ppm or 100 ppm CO impaired cytochrome c release, caspase-3 activation, and apoptosis in neocortex and hippocampus of 10 day old CD-1 mice. CO increased NeuN protein, neuronal numbers, and resulted in megalencephaly. CO-exposed mice demonstrated impaired memory and learning and reduced socialization following exposure. Thus, CO-mediated inhibition of neuroapoptosis might represent an important etiology of acquired neurocognitive impairment and behavioral disorders in children.     

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII.

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.     

Proliferation of Antibiotic-Producing Bacteria and Concomitant Antibiotic Production as the Basis for the Antibiotic Activity of Jordan's Red Soils

Anecdotes, both historical and recent, recount the curing of skin infections, including diaper rash, by using red soils from the Hashemite Kingdom of Jordan. Following inoculation of red soils isolated from geographically separate areas of Jordan, Micrococcus luteus and Staphylococcus aureus were rapidly killed. Over the 3-week incubation period, the number of specific types of antibiotic-producing bacteria increased, and high antimicrobial activity (MIC, ∼10 μg/ml) was observed in methanol extracts of the inoculated red soils. Antibiotic-producing microorganisms whose numbers increased during incubation included actinomycetes, Lysobacter spp., and Bacillus spp. The actinomycetes produced actinomycin C₂ and actinomycin C₃. No myxobacteria or lytic bacteriophages with activity against either M. luteus or S. aureus were detected in either soil before or after inoculation and incubation. Although protozoa and amoebae were detected in the soils, the numbers were low and did not increase over the incubation period. These results suggest that the antibiotic activity of Jordan''s red soils is due to the proliferation of antibiotic-producing bacteria.There is a growing recognition of the pressing need for new antimicrobial agents for the treatment of infectious diseases (11, 38). As just one cogent example, new antibiotics are in high demand for the treatment of Staphylococcus aureus infections (25), particularly due to the emergence of methicillin-resistant S. aureus in communities and hospitals (25, 41). In addition, providing effective and affordable antibiotics to people in epidemic-prone developing countries remains a major challenge (37).Historically, natural products have played a key role in the discovery and development of many antibiotics (34). In particular, soil-based actinomycetes have been the source of countless drugs, such as streptomycin, actinomycin, erythromycin, and vancomycin, to name only a few (18). One approach to the discovery of new antimicrobial agents from natural sources has been to use folklore or historical records to guide the collection of samples (20).Through our ongoing studies of the biodiversity of the Hashemite Kingdom of Jordan (Jordan) (2-7, 35, 36), we were intrigued by anecdotes of the antibiotic-like properties of red soil, used historically for treating skin infections and diaper rash and still in use in some communities as an inexpensive alternative to pharmaceutical products. Within Jordan, there are four major biogeographic regions (8, 21), and red soils are most commonly found in the Mediterranean region of the northwestern portion of Jordan, near cities such as Irbid, Ramtha, and Ajloun. An area away from housing, preferably not touched by feet and thus considered clean, is chosen, and soil below the surface is collected, as the surface is considered contaminated or not clean. After the infected area of skin is washed and dried, the sieved soil is applied daily as either a powder or paste until the infection subsides. The basis for the antimicrobial activity of red soils is not known.Antimicrobial activity of soils against inoculated microorganisms has been attributed to abiotic or biotic factors. Abiotic activity has been shown to be responsible for the antimicrobial activities of clay minerals used in the treatment of a mycobacterial skin infection known as Buruli ulcer (28). Soil texture was found to influence the survival of Pseudomonas fluorescens and Bacillus subtilis in soil (40), while soil temperature and pH and the presence of roots affected the leaching of a genetically modified strain of P. fluorescens in soil (29). Biotic factors, including predation and antimicrobial-producing or lytic microorganisms, were suggested as mechanisms for killing microorganisms introduced into soils (1, 14, 31, 32). Prior inoculation of soil with one strain of P. fluorescens reduced the ability of a second P. fluorescens strain to colonize (19). A phenazine pigment produced by a P. fluorescens strain was shown to be responsible for biological control of a root disease of wheat caused by Gaeumannomyces graminis var. tritici (39), and it has been shown that filaments of the biocontrol fungus Trichoderma grow toward fungal pathogens and release antibiotics and lytic enzymes (9). With that background as a guide, we undertook an investigation to identify the basis for the antimicrobial activity of Jordan''s red soils.     

ERp27, a new non-catalytic endoplasmic reticulum-located human protein disulfide isomerase family member, interacts with ERp57

Protein folding and quality control in the endoplasmic reticulum are critical processes for which our current understanding is far from complete. Here we describe the functional characterization of a new human 27.7-kDa protein (ERp27). We show that ERp27 is a two-domain protein located in the endoplasmic reticulum that is homologous to the non-catalytic b and b' domains of protein disulfide isomerase. ERp27 was shown to bind Delta-somatostatin, the standard test peptide for protein disulfide isomerase-substrate binding, and this ability was localized to the second domain of ERp27. An alignment of human ERp27 and human protein disulfide isomerase allowed for the putative identification of the peptide binding site of ERp27 indicating conservation of the location of the primary substrate binding site within the protein disulfide isomerase family. NMR studies revealed a significant conformational change in the b'-like domain of ERp27 upon substrate binding, which was not just localized to the substrate binding site. In addition, we report that ERp27 is bound by ERp57 both in vitro and in vivo by a similar mechanism by which ERp57 binds calreticulin.     

Substrate recognition by the protein disulfide isomerases

Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds. Their primary function is to stabilize the folded structure of the protein, although disulfide bond formation can also play a regulatory role. Native disulfide bond formation is not trivial, so it is often the rate-limiting step of protein folding both in vivo and in vitro. Complex coordinated systems of molecular chaperones and protein folding catalysts have evolved to help proteins attain their correct folded conformation. This includes a family of enzymes involved in catalyzing thiol-disulfide exchange in the endoplasmic reticulum, the protein disulfide isomerase (PDI) family. There are now 17 reported PDI family members in the endoplasmic reticulum of human cells, but the functional differentiation of these is far from complete. Despite PDI being the first catalyst of protein folding reported, there is much that is still not known about its mechanisms of action. This review will focus on the interactions of the human PDI family members with substrates, including recent research on identifying and characterizing their substrate-binding sites and on determining their natural substrates in vivo.     

Widespread Distribution of Poribacteria in Demospongiae

Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.Recently, a novel bacterial phylum, termed “Poribacteria,” was discovered, and members of this phylum have been found exclusively in sponges (2). Phylogenetic analyses of 16S rRNA genes indicated that poribacteria are evolutionarily deeply branching organisms and related to a superphylum composed of Planctomycetes, Verrucomicrobia, and Chlamydia (11). Poribacterial 16S rRNA genes contain 13 of 15 planctomycete signature nucleotides, but a level of sequence divergence of more than 25% compared to any other bacterial phylum, including the Planctomycetes, justifies the status of this taxon as an independent phylum. A consistent treeing pattern is difficult to resolve in comparative phylogenetic sequence analyses, making the poribacteria an unusual line of phylogenetic descent. In addition to their divergent status as a separate phylum on the basis of the 16S rRNA sequence, poribacteria are also divergent because they may have a compartmentalized cell structure, a cell plan they share only with members of the phyla Planctomycetes and Verrucomicrobia (2). They are also of interest for understanding the potential contribution of obligate sponge-associated bacteria to the sponges harboring them and as an example of a yet-to-be-cultured group of bacteria associated with invertebrate tissue apparently exclusively but for unknown reasons. This study aimed to further explore the presence and diversity of poribacteria in different marine demosponge genera using samples from around the world.The Mediterranean sponges were collected by scuba divers offshore at Banyuls sur Mer, France (42°29′N, 03°08′E). The Caribbean sponges were collected offshore at Little San Salvador Island, Bahamas (24°32′N, 75°55′W). The eastern Pacific sponge Aplysina fistularis was collected offshore at San Diego, CA (32°51′N, 117°15′W). The western Pacific sponge Theonella swinhoei was collected offshore at Palau (07°23′N, 134°38′E). All non-Great Barrier Reef (non-GBR) sponges were collected between May and July 2000, and once individual sponge specimens were brought to the surface, they were frozen in liquid nitrogen on board ship and stored at −80°C until microbiological processing (9). The GBR marine sponges were collected off Heron Island Research Station (23°27′S, 151°5′E) in April 2002 (5). Pseudoceratina clavata was collected by scuba divers at a depth of 14 m, and Rhabdastrella globostellata was collected at a depth of ca. 0.5 m after a reef walk consisting of a few hundred meters. The samples were immediately placed in plastic bags and brought to Heron Island Research Station, where they were stored at −80°C until processing. Sponge DNA was extracted as described previously (2, 5).Total sponge-derived genomic DNA was screened by PCR for the presence of poribacteria using a 16S rRNA gene primer set. Poribacterial 16S rRNA genes were amplified by employing a pair of Poribacteria-specific primers, POR389f (5′-ACG ATG CGA CGC CGC GTG-3′) and POR1130r (5′-GGC TCG TCA CCA GCG GTC-3′) (2). The poribacterial PCR products that were ca. 740 bp long derived from one sponge individual were cloned into the pGEM-T Easy vector (Promega, Madison, WI). Clone inserts were digested with restriction endonucleases MspI and HaeIII (New England Biolabs, Inc., United States), characterized to obtain restriction profiles and unique profiles, and sequenced. The compiled partial 16S rRNA gene sequences were then analyzed using BLASTN to select the most closely related poribacterial reference sequences.The sequences exhibiting levels of similarity of less than 97% were used for further analysis. Poribacterial 16S rRNA gene sequences were aligned using the ARB software package (7). The resulting alignment was imported into PAUP (10) and analyzed by using distance, maximum parsimony, and maximum likelihood algorithms together with bootstrap resamplings (3,000, 3,000, and 200 resamplings, respectively), and the resulting bootstrap values were applied to nodes on the ARB neighbor-joining tree. Signature sequences were detected using the ARB software package. A signature sequence is defined here as a short sequence that is present in a group of poribacterial sequences in a phylogenetic clade but is not found in any other clade in the poribacterial tree.Analysis of the 16S rRNA gene clone library sequences generated from sponge tissues revealed the presence of poribacteria in sponge individuals belonging to the orders Verongida, Astrophorida, Dictyoceratida, Haplosclerida, Lithistida, and Homosclerophorida, while poribacteria could not be detected in sponges belonging to the orders Hadromerida and Agelasida. In the order Halichondrida, poribacteria were detected in Xestospongia muta but not in Haliclona sp. Altogether, nine sponge species were added to the list of Poribacteria-containing sponges (Table ​(Table1).1). Three distinct clades were observed that were clearly supported by bootstrap values greater than 75 with every tree-building algorithm applied (Fig. ​(Fig.1),1), and one clade (clade I) was supported by bootstrap values of 64, 98, and 71 in distance, maximum parsimony, and maximum likelihood trees, respectively. Similarity calculations using approximately 740-bp amplified poribacterial 16S rRNA gene fragments and other poribacterial sequences from the NCBI database showed that the dissimilarity between clades was consistent with their separation in phylogenetic trees. For example, the levels of dissimilarity between members of clade I and clade II were 3 to 8%, while the levels of dissimilarity between members of clades I and III and between members of clades I and IV were 10 to 14% and 11 to 15%, respectively.Open in a separate windowFIG. 1.Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing relationships of poribacterial clones from different global regions. The poribacterial clones on the right are additional clones belonging to the same clades as strains in the tree at the same level. Bootstrap confidence values of >75% for distance, maximum parsimony, and maximum likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba; C, Aplysina cavernicola; F, Aplysina fistularis; L, Aplysina lacunosa; S, Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella globostellata; T, Theonella swinhoei; X, Xestospongia muta. Scale bar = 0.1 nucleotide substitution per site.

TABLE 1.

Distribution of poribacteria in different demosponge orders