Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa.

Following the discovery of the first Eukarya in the deep subsurface, intense interest has developed to understand the diversity of eukaryotes living in these extreme environments. We identified that Platyhelminthes, Rotifera, Annelida and Arthropoda are thriving at 1.4 km depths in palaeometeoric fissure water up to 12,300 yr old in South African mines. Protozoa and Fungi have also been identified; however, they are present in low numbers. Characterization of the different species reveals that many are opportunistic organisms with an origin due to recharge from surface waters rather than soil leaching. This is the first known study to demonstrate the in situ distribution of biofilms on fissure rock faces using video documentation. Calculations suggest that food, not dissolved oxygen is the limiting factor for eukaryal population growth. The discovery of a group of Eukarya underground has important implications for the search for life on other planets in our solar system.

The effect of different treatment modalities on the calcification potential and cross-linking stability of bovine pericardium.

Porcine heart valves and bovine pericardium exhibit suitable properties for use as substitutes in cardiothoracic surgery, but must meet several requirements to be safe and efficient. Treatment with glutaraldehyde (GA) render some of these requirements, but calcification and degradation post-implant remain a problem. This study aimed to identify additional biochemical treatments that will minimize calcification potential without compromising the physical properties of pericardium. Pericardium treated with GA calcified severely after 8 weeks in the subcutaneous rat model, compared to tissue treated with higher concentrations of glycosaminoglycans (GAG) and commercial Glycar patches. GA, lower concentrations GAG and Glycar pericardium had high denaturation temperatures due to enhanced cross-linking. Tensile strength of GA tissue was significantly lower than GAG-treated or Glycar tissues, due to lower water content with resultant lower flexibility and suppleness. Pericardium treated with 0.01 M GAG gave acceptable denaturation temperatures, tensile strength and reduced calcification potential. All tissue treatments evoked comparable host immune responses, and no significant difference in resistance to enzymatic degradation. Ineffective stabilization and fixation of cross-links following GAG treatment, as well as limited penetration into the pericardium, resulted in GAG leaching out into the surrounding host tissue or storage medium, and prohibits safe clinical use of such tissue.

Nematoda from the terrestrial deep subsurface of South Africa.

Since its discovery over two decades ago, the deep subsurface biosphere has been considered to be the realm of single-cell organisms, extending over three kilometres into the Earth's crust and comprising a significant fraction of the global biosphere. The constraints of temperature, energy, dioxygen and space seemed to preclude the possibility of more-complex, multicellular organisms from surviving at these depths. Here we report species of the phylum Nematoda that have been detected in or recovered from 0.9-3.6-kilometre-deep fracture water in the deep mines of South Africa but have not been detected in the mining water. These subsurface nematodes, including a new species, Halicephalobus mephisto, tolerate high temperature, reproduce asexually and preferentially feed upon subsurface bacteria. Carbon-14 data indicate that the fracture water in which the nematodes reside is 3,000-12,000-year-old palaeometeoric water. Our data suggest that nematodes should be found in other deep hypoxic settings where temperature permits, and that they may control the microbial population density by grazing on fracture surface biofilm patches. Our results expand the known metazoan biosphere and demonstrate that deep ecosystems are more complex than previously accepted. The discovery of multicellular life in the deep subsurface of the Earth also has important implications for the search for subsurface life on other planets in our Solar System.

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library.

AIMS: The aim of this study was to demonstrate the application of environmental sample pre-enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s). METHODS AND RESULTS: A positive recombinant clone (UVCL29), conferring an esterase phenotype was identified from a shotgun gene library. The complete sequence of the 3.0 kb DNA insert from the pUVCL29 recombinant plasmid was obtained using primer-walking strategies. Nucleotide sequence analysis revealed a complete 945 bp open reading frame (ORF1). Translational analysis of the ORF1 showed a protein of 314 amino acids (named EstAM) with a predicted molecular weight of 34 kDa. EstAM's primary structure showed a classical (-G-D-S-A-G-) motif, corresponding with the generally conserved (G-x-S-x-G) esterase signature motif. Identity searches indicated that EstAM has high sequence similarity with esterases from family IV. EstAM was successfully expressed in Escherichia coli in a biologically active form. Partial purification was achieved using a one-step Pro-PurTM IMAC column. Biochemical characterization revealed that EstAM has a temperature optimum of 40 degrees C. CONCLUSION: Based on its substrate profile, EstAM was classified as a carboxylesterase because of its preference for short p-nitrophenyl ester substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is a demonstration of the successful application of environmental sample pre-enrichment technology in accessing novel esterases from a mining environment.

Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases.

The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures.

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent kS value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h(-1), with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h(-1), but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h(-1), which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.

Purification and characterization of a metalloprotease from Chryseobacterium indologenes Ix9a and determination of the amino acid specificity with electrospray mass spectrometry.

The heat-stable protease from Chryseobacterium indologenes Ix9a was purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction.

Effect of lipoproteins on the differentiation of 3T3-L1 and human preadipocytes in cell culture.

High-density and low-density lipoprotein (LDL) stimulated 3T3-L1 and human preadipocyte differentiation in vitro. In both cell types, LDL exhibited the greatest stimulatory effect. LDL suppressed the development of catecholamine-stimulated lipolysis in differentiating 3T3-L1 and human preadipocytes when present during preadipocyte proliferation and early stages of differentiation. The effect of lipoproteins on development of human preadipocyte (but not 3T3-L1) lipolysis may involve beta-adrenoceptors.

Geotrichum candidum P-5 produces an intracellular serine protease resembling chymotrypsin.

A wide range of intra- and extracellular microbial proteases has been studied and characterized. These enzymes are mostly extracellular and in some cases they may resemble 'classical' serine proteases. As part of a programme in which the lipase and protease activities of the fungus Geotrichum candidum are being studied, an intracellular protease with an apparent chymotrypsin-like specificity was detected. The serine protease was isolated from biomass using ion-exchange and exclusion chromatography. Kinetic characterization was done using a series of synthetic substrates and inhibitors. Aprotinin-sepharose affinity chromatography was used to isolate a fraction for molecular size determination on SDS-PAGE. The purified protease, which could hydrolyse haemoglobin as protein substrate, was obtained with a 30-fold purification and a yield of 44%, but it was very unstable and rapidly lost activity. The enzyme which bound to the affinity column had a single subunit mass of 278 kDa. Kinetic analysis showed a similarity with trypsin and chymotrypsin, but tending more towards chymotrypsin in that a bulky aromatic group, e.g. phenylalanine in the P1 position, was preferred. The optimum pH was in the region of 7-8.25. Inhibition patterns indicated that the enzyme was a serine protease with no metal dependence, although it was stabilized by magnesium ions. The enzyme seems to share some properties with other intra- and extracellular microbial serine proteases. The exact function of the enzymatic activity is still unclear, but it is suggested that it may be involved with intracellular protein turnover.

Isolation and characterization of adipose tissue glycerol-3-phosphate dehydrogenase.

Recent studies on the differentiation of human preadipocytes have extensively used GPDH as a convenient marker enzyme to follow the development of the cells. Since the properties of human adipose tissue GPDH is largely unknown, it was considered of interest to characterize the purified enzyme. Glycerol-3-phosphate dehydrogenase was purified to homogeneity using blue Sepharose and hydroxylapatite chromatography. Monomeric molecular mass of GPDH was estimated using SDS-PAGE while the dimeric mass was estimated using non-denaturing PAGE. Fluorometric titrations were used to measure the binding of NADH to the enzyme. Inactivation experiments with proteolytic enzymes, urea and heat treatment were used to investigate a possible conformational change due to NADH binding. The purified enzyme displayed a monomeric molecular mass of 35,000 Da, a dimeric molecular mass of 74,000 Da and an isoelectric point (pI) of 5.85. The enzyme exhibited a pH optimum of 7.5 for the reduction of DHAP and 9.0 for G-3-P oxidation. Glycerol (50%) was found to stabilize the enzyme activity during storage, but altered the kinetic properties of the enzyme, acting as a competitive activator with respect to DHAP reduction. GPDH was inhibited by sulfhydryl modifying reagents and fatty acids. The effectiveness of inhibition by saturated fatty acids increased proportionately with chain length from decanoate to stearate. In addition preincubation of the enzyme, in the presence of oleate, resulted in a time dependent inactivation. Time dependent inactivation of GPDH by both iodoacetate and oleate was prevented by the presence of NADH but not NAD+.(ABSTRACT TRUNCATED AT 250 WORDS)

Catecholamine stimulated lipolysis in differentiated human preadipocytes in a serum-free, defined medium.

Adipocyte precursors from the stromal vascular fraction of human adipose tissue were allowed to differentiate in serum-free defined medium, whereafter their catecholamine stimulated lipolytic response was compared to that of mature isolated human adipocytes. Seventy-five to ninety percent of the fibroblast-like cells accumulated lipid droplets and glycerol-3-phosphate dehydrogenase activities of 1,000-2,800 mU/mg protein were measured in cell homogenates of differentiated cells. Lipolysis could be stimulated by both isoproterenol and norepinephrine in both differentiated preadipocytes as well as mature adipocytes. The results obtained with beta-adrenergic agents suggested the presence of a higher affinity receptor in differentiated preadipocytes as compared to mature adipocytes. Mature adipocytes responded well to beta-adrenergic agents, but no antilipolytic alpha 2-adrenergic response was observed in the differentiated preadipocytes. The presence of Gi proteins in the differentiated preadipocytes was suggested by the antilipolytic effect of adenosine as well as the lipolytic activity generated by pertussis toxin. In conclusion, our medium supported the differentiation of a very high percentage of human preadipocytes which developed a sensitive beta-adrenergic lipolytic response but which lacked an alpha 2-adrenergic antilipolytic response.

Mitochondrial and peroxisomal fractions derived from human white adipocytes.

1. A procedure is described for the separation of intact peroxisomes from human white adipocytes using a linear metrizamide gradient (20-50% w/v). 2. Peroxisomes were found in the high density region of the gradient in an intact form. 3. Mitochondria were distributed in the high density and low density regions of the gradient. 4. Lysosomes separated well from the peroxisomes, occurring only in the low density region of the gradient. 5. Low levels of glyoxylate cycle enzyme activities (isocitrate lyase and malate synthase) were detected within the light and heavy mitochondrial pellet fractions.

The production of the ostrich NH2-terminal POMC fragment requires cleavage at a unique signal peptidase site.

The NH2-terminal fragment of ostrich proopiomelanocortin was isolated and purified following acid/acetone extraction. The amino acid sequence was deduced by automatic Edman degradation of the native as well as CNBr-, tryptic-, and S. aureus protease-derived peptides. Primary structure analysis reveals its close resemblance to other known sequences, especially to amphibian POMC. The usual Trp/Gln-Cys NH2-terminal sequence found in all other homologous sequences, is replaced here by an His-Gly-Pro-Cys sequence. In addition, the gamma-MSH sequence, contrary to salmon POMC, is present and contains three substitutions, namely a Ser, an Asn, and a Lys residue substituting the normally occurring mammalian Gly, Asp, and Arg residue, respectively. Finally, the molecular weight of this fragment as deduced from ion-spray mass spectrometry and sedimentation equilibrium centrifugation is in close agreement with the proposed structure.

The effect of high concentrations of alpha 2-adrenoceptor antagonists on the lipolytic responsiveness of isolated human adipocytes.

1. Non-specific effects of alpha 2-adrenergic antagonists on human adipocyte lipolysis are reported, and revealed that yohimbine and RX-821002 (10(-4) M) decreased the maximum lipolytic response and increased the ED50 towards norepinephrine and isoproterenol. 2. These effects were not mediated by adenosine receptors, alpha 2-adrenoceptors or Gi proteins. 3. Whereas alpha 2-antagonists, even as low as 10(-8) M, decreased the binding affinity of [3H]CGP-12177, levels as high as 10(-3) M of these antagonists were needed to cause 10-20% displacement of the beta-selective antagonist. 4. We propose that the antilipolytic effects of alpha 2-antagonists can only partly be explained by non-specific binding to beta-adrenoceptors, and that some other mechanism is also involved.

The isolation and partial characterization of catalase and a peroxidase active fraction from human white adipose tissue.

1. A procedure is described for the purification of catalase and a peroxidase active fraction from human white adipose tissue. 2. Gel electrophoresis on SDS-PAGE revealed relative molecular masses of 202,900 and 208,600 for the active catalase and peroxidase molecules respectively (nonreducing conditions), as compared to 56,800 and 49,800 for the monomers under reducing conditions, thus indicating the likelihood of tetramers in the intact state. 3. The two purified enzymes differ with regard to pH optima (5-9 for catalase and 3 for peroxidase), temperature stability (up to 50 degrees C for catalase and 70 degrees C for peroxidase) and Km values towards H2O2 (38.9 mM for catalase and 7.69 mM for peroxidase, which was also active in oxidizing a number of o-dihydricphenols as second substrates). 4. The catalase enzyme showed uncompetitive inhibition by the irreversible inhibitor 3-amino-1,2,4-triazole (AT), Ki = 5.4 mM.

Partial characterisation of human and porcine adipose acidic protease activity.

1. An aspartic protease was isolated from human and porcine white adipose tissue and from isolated human adipocytes. The three preparations appeared to represent the same enzyme. 2. Electrofocusing of all three preparations revealed two bands corresponding to a pI of 3.6 and 4.4. respectively. On PAGE a single band in the same position was obtained in all three cases. 3. Both the porcine and human fractions were optimally active at pH 3.4, using acid denatured haemoglobin as substrate, and both activities were strongly inhibited by pepstatin and iodoacetate. 4. The Km values for haemoglobin for the porcine and human proteases were 0.16 and 0.14 mM respectively, whereas Vmax values of 30 and 33 units.nmol-1, respectively, were obtained.

The primary culture of mouse adipocyte precursor cells in defined medium.

1. A defined medium supporting the proliferation and differentiation of adipocyte precursors isolated from inguinal fat pads of 8-12-day-old mice was developed. 2. It consists of a 1:1 mixture of DME and WAJC404A media supplemented with insulin (10 micrograms/ml), transferrin (10 micrograms/ml), fibroblast growth factor (10 ng/ml) and high density lipoproteins (HDL) (90 micrograms protein/ml). 3. DME-F12 medium (1:1 mixture) used as a nutrient mixture in the defined medium of rat and human adipocyte precursors was inadequate for cultivating mouse adipocyte precursors. 4. HDL had a definite beneficial effect on both preadipocyte growth and differentiation. 5. Differentiation was enhanced by addition of dexamethasone (10(-9) M) but could be almost completely inhibited by transforming growth factor beta 1 (TGF-beta 1). 6. TGF-beta 1 was shown to be effective only when present in the early stages of differentiation.

Purification and primary structure of glucagon from ostrich pancreas splenic lobes.

Glucagon is a highly conserved polypeptide hormone which appears to play a more important role in regulation of glycaemia in birds than insulin. Ostrich glucagon was isolated and purified from ostrich pancreas splenic lobes using an adapted acid ethanol extraction procedure, gel filtration, ion exchanges, and HPLC steps. The purified glucagon fraction appeared to contain small quantities of a more acidic contaminant (polyacrylamide gel isoelectric focussing, PAGE) but appeared homogeneous on SDS-PAGE. Amino acid analysis and sequence analysis showed identity with the duck hormone. Identity with the duck hormone was confirmed by liquid phase as well as gas phase sequencing. The ostrich glucagon preparation seemed to have a higher Km than the porcine homologue in stimulating glycerol release from isolated chicken adipocytes.

Properties of the isoenzyme forms A-1, A-2 and B of N-acetyl-beta-D-glucosaminidase purified from baboon kidneys.

Three forms of N-acetyl-beta-D-glucosaminidase (NAG: A, B and I) were separated from baboon kidney using Con A-Sepharose and DEAE-Trisacryl chromatography. 2. The A form was further purified into two forms A-1 and A-2 using hydroxylapatite chromatography and anodic PAGE. Both were homogeneous on SDS-PAGE and anodic PAGE but microheterogeneous on PAG-IEF, which could be eliminated by prior treatment with endoglycosidase H or glycopeptidase F. 3. The carbohydrate content accounted for some of this microheterogeneity since it varied from 31 for A-1 to 17% for A-2 and the sialic acid was 6 and 1%. Deamidation may also contribute since the acidic amino acids (29 mol%) and ammonia were high following acid hydrolysis. 4. The mol. wt for A-1, determined by SDS-PAGE, was 52.1 K. 5. The pH optimum was 4.55 and the pI4.97. 6. The optimum temperature for NAG A and B was 50 degrees and 42 degrees C, but B retained more activity above 55 degrees C. 7. The Km for N-acetyl-beta-D-glucosamine and -galactosamine for both isoforms was 0.497 and 0.627 mM respectively. 8. Several ions were found to be uncompetitive inhibitors. Ag+ and Pb2+ were the most potent having Ki values of 3.6 and 8.5 mM respectively. Acetate acted as a competitive inhibitor.

The ostrich pituitary contains a major peptide homologous to mammalian chromogranin A(1-76).

A major peptide related to the NH2-terminal fragment (position 1 to 76) of mammalian chromogranin A was isolated from ostrich adenohypophyses following acid-acetone extraction. The complete amino acid sequence of the homogenous peptide was deduced following automatic Edman degradation of the native peptide as well as of CNBr-, tryptic- and Lysobacter-derived peptides. The 76 amino acid sequence is strikingly homologous to bovine (80.3% sequence identity), porcine (79.0%), human (79.0%) and rat (72.4%) corresponding sequences, but much less so to human chromogranin B (22.4%). As this peptide is followed in bovine, porcine and human structure by a pair of basic residues (Lys-Lys), it could conceivably be produced during maturation in secretory granules. Finally, its structure appears to contain two potential amphipathic helices joined by the single disulfide bridge present in all chromogranin A and B molecules.