Global harmonization of vaccine testing requirements: Making elimination of the ATT and TABST a concrete global achievement.

This one-day symposium organized by Humane Society International (HSI) brought together 18 international experts from Argentina, Brazil, China, Europe, India, Russia, South Africa and the United States to discuss the elimination of the abnormal toxicity test (ATT) from the testing requirements for human vaccines as well as the target animal batch safety test (TABST) and the laboratory animal batch safety test (LABST) for veterinary vaccines. Participants reported on country-specific regulatory requirements and, where present, the perspectives on waiver and elimination of those tests. In addition, the attendees, with HSI in the role of facilitator, moved to define the barriers to the complete elimination or waiving of these tests.

Genetic determinant of Bacillus pumilus lipase lethality and its application as positive selection cloning vector in Escherichia coli.

Positive selection vectors carry genes that upon expression produce proteins that cause host cell deaths. Insertion of foreign DNA fragments within the ORF of the gene disrupts the lethal effect of the expressed protein. This study described the cloning of Family I.

Whole Genome Comparison of Thermus sp. NMX2.A1 Reveals Principle Carbon Metabolism Differences with Closest Relation Thermus scotoductus SA-01.

Genome sequencing of the yellow-pigmented, thermophilic bacterium Thermus sp. NMX2.A1 resulted in a 2.

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.

Deep subsurface mine stalactites trap endemic fissure fluid Archaea, Bacteria, and Nematoda possibly originating from ancient seas.

Stalactites (CaCO3 and salt) from water seeps are frequently encountered in ceilings of mine tunnels whenever they intersect water-bearing faults or fractures. To determine whether stalactites could be mineralized traps for indigenous fracture water microorganisms, we analyzed stalactites collected from three different mines ranging in depth from 1.3 to 3.

Analysis of genomic rearrangements, horizontal gene transfer and role of plasmids in the evolution of industrial important Thermus species.

Background: Bacteria of genus Thermus inhabit both man-made and natural thermal environments. Several Thermus species have shown biotechnological potential such as reduction of heavy metals which is essential for eradication of heavy metal pollution; removing of organic contaminants in water; opening clogged pipes, controlling global warming among many others. Enzymes from thermophilic bacteria have exhibited higher activity and stability than synthetic or enzymes from mesophilic organisms.

Analysis of protein thermostability enhancing factors in industrially important thermus bacteria species.

Elucidation of evolutionary factors that enhance protein thermostability is a critical problem and was the focus of this work on Thermus species. Pairs of orthologous sequences of T. scotoductus SA-01 and T.

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.

Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01.

Background: Many strains of Thermus have been isolated from hot environments around the world. Thermus scotoductus SA-01 was isolated from fissure water collected 3.2 km below surface in a South African gold mine.

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.

Crystal structure of a thermostable old yellow enzyme from Thermus scotoductus SA-01.

Recent characterization of the chromate reductase (CrS) from the thermophile Thermus scotoductus SA-01 revealed this enzyme to be related to the Old Yellow Enzyme (OYE) family. Here, we report the structure of a thermostable OYE homolog in its holoform at 2.2A as well as its complex with p-hydroxybenzaldehyde (pHBA).

A thioredoxin reductase-like protein from the thermophile, Thermus scotoductus SA-01, displaying iron reductase activity.

The transition metal iron is an important element for the sustenance of life--it can function either as an electron acceptor or as a donor and serves as a cofactor in many enzymes activities. The cytoplasmic NAD(P)H-dependent ferric reductase in Thermus scotoductus SA-01 shares high sequence and structural similarity to prokaryotic thioredoxin reductases. Here we report the sequence of the ferric reductase (which is typically annotated as a thioredoxin reductase-like protein) and a comparative kinetic study with the thioredoxin reductase from SA-01.

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.

Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species.

Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky-and blunt-end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers.

In vitro effect of a thrombin inhibition peptide selected by phage display technology.

A repeated selection of phages from a cyclic heptapeptide phage display library resulted in the enrichment of phages that bind to human alpha-thrombin. One clone of the binding phages that competed with PPACK for binding to thrombin and that had the best binding characteristics was chosen. The amino acid sequence of the peptide displayed on this phage was determined and a peptide with the sequence, Cys-Asn-Arg-Pro-Phe-Ile-Pro-Thr-Cys was synthesised.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.