An alternative sterility assessment for parenteral drug products using isothermal microcalorimetry.

AIMS: Production and release of injectable drug solutions are highly regulated since the administration of injectables bypasses natural body barriers. The sterility test is the last opportunity of product quality assessment. However, sterility is currently assessed by visual inspection (VI) that is time consuming and somewhat subjective. Therefore, we assessed isothermal microcalorimetry (IMC) as a replacement for the VI of the filtration based state-of-the-art sterility control. METHODS AND RESULTS: We used ATCC strains and house isolates to artificially contaminate frequently produced monoclonal antibodies (Avastin, Mabthera, Herceptin). After filtration, growth was assessed with IMC. Growth of all micro-organisms was reliably and reproducibly detected 4 days after inoculation, which was significantly faster than with VI. CONCLUSIONS: The reliability and the sensitivity of IMC have a large potential to improve sterility controls. Further evaluation of this alternative method is therefore highly recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: Drug safety is of great concern for public health. Faster and safer drug production could be achieved using the technique described here. All the tests were performed with real manufactured drugs and complied with pharmaceutical standards. This suggests that drug sterility testing can be improved with potentially increased safety and cost reduction.

Influence of epigallocatechin gallate and phenolic compounds from green tea on the growth of Oenococcus oeni.

AIMS: To investigate the effect of phenolic compounds on the growth of Oenococcus oeni. METHODS AND RESULTS: Oenococci are usually grown in media often supplemented with complex additives such as tomato juice. In order to improve our knowledge about the growth requirements of oenococci, we added several juices and leaf extracts such as green tea to the culture media and screened them for growth-stimulating substances to substitute complex supplements such as juices by more defined components. We found that also green tea could cause a growth stimulation of Oenococcus oeni strain B2. CONCLUSIONS: Further experiments showed that the stimulating effect was as a result of the phenolic compounds of green tea, especially epigallocatechin gallate (EGCG). On the other hand, EGCG could also inhibit the growth of O. oeni strain B2 just depending on its concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: Individual catechins should have a minor influence on the growth of oenococci during wine making as their concentration in grapes is <30 mg kg(-1) grape. Whether there is a synergistic effect of the different catechins in wine has to be investigated.

Putative sigma factor SigI (YkoZ) of Bacillus subtilis is induced by heat shock.

A Bacillus subtilis disruption mutant with a mutation in sigI (formerly ykoZ) shows a temperature-sensitive growth on agar plates. The transcription of the sigI gene is heat shock induced in rich medium but not in minimal medium. Proteome studies revealed a reduced amount of GsiB protein in the sigI mutant under heat shock conditions.

The genes of lepA and hemN form a bicistronic operon in Bacillus subtilis.

The IepA operon of Bacillus subtilis was found to be bicistronic and to consist of the two genes IepA and hemN, which encode a putative GTP-binding protein and an oxygen-independent coproporhyrinogen III oxidase, respectively. The IepA operon is located immediately upstream of the dnaK operon. Both operons are transcribed in the same direction and are not separated by an obvious transcription-terminator-like structure. The IepA operon is preceded by a potential vegetative promoter, and there is a putative strong intergenic terminator between IepA and hemN. Northern blot experiments revealed only a transcript corresponding to IepA, but expression of hemN was demonstrated in slot-blot and immunoblot experiments using antibodies raised against His-tagged HemN. The data suggest that most of the transcripts originating at the potential vegetative promoter are terminated at the intergenic terminator. Readthrough transcription into the downstream dnaK operon was not found.

CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis.

The dnaK and groESL operons of Bacillus subtilis are preceded by a potential sigma 43 promoter sequence (recognized by the vegetative sigma factor) and by an inverted repeat (IR) consisting of 9 bp separated by a 9-bp spacer. Since this IR has been found in many bacterial species, we suspected that it might be involved in heat shock regulation. In order to test this hypothesis, three different mutational alterations of three bases were introduced within the IR preceding the dnaK operon. These mutations were crossed into the chromosome of B. subtilis, and expression of the dnaK and of the unlinked groESL operons was studied. The dnaK operon exhibited increased expression at low temperature and a reduction in the stimulation after temperature upshift. Furthermore, these mutations reduced expression of the groESL operon at low temperature by 50% but did not interfere with stimulation after heat shock. These experiments show that the IR acts as a negative cis element of the dnaK operon. This conclusion was strengthened by the observation that the IR reduced expression of two different transcriptional fusions significantly after its insertion between the promoter and the reporter gene. Since this IR has been described in many bacterial species as preceding only genes of the dnaK and groESL operons, both encoding molecular chaperones (39 cases are documented so far), we designated this heat shock element CIRCE (controlling IR of chaperone expression). Furthermore, we suggest that this novel mechanism is more widespread among eubacteria than the regulation mechanism described for Escherichia coli and has a more ancient origin.

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis.

By using an internal part of the dnaK gene from Bacillus megaterium as a probe, a 5.2-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf39-grpE-dnaK-dnaJ. orf39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orf39 (S1 and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in alpha-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orf39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 223 degrees on the B. subtilis genetic map.

Tn5cos: a transposon for restriction mapping of large plasmids using phage lambda terminase.

A method for the rapid restriction mapping of large plasmids has been developed. A 400-bp fragment of phage lambda DNA containing the cos region has been inserted into Tn5. After in vivo transposition of this Tn5cos element into the plasmid of choice, the plasmid is isolated and linearized at its cos site with phage lambda terminase (Ter). Such Ter linearization was about 70% efficient. After partial digestion of the linear molecules with the appropriate restriction enzyme, the products are selectively labelled at the right or left cohesive phage lambda DNA termini by hybridization with digoxygenin (DIG)-11-dUTP-labelled (using terminal transferase) oligodeoxyribonucleotides complementary to the single-stranded cos ends. After pulsed field gel electrophoresis, the labelled fragments are visualized in the dried gel using a DIG-detection kit. The restriction map can be directly determined from the 'ladder' of partial digestion products.

Iron stores of liver, spleen and bone marrow, and serum iron concentrations in female dairy cattle in relationship to age.

Storage iron in spleen, liver and bone marrow of cattle significantly increased after three years of age, reached mximal concentrations at five years and remained constant until nine years of age. Storage iron concentrations were always highest in the spleen. In liver and bone marrow much lower concentrations were found. In contrast to storage iron, serum iron remained relatively constant between one and eight years of age.