Environmentally constrained mutation and adaptive evolution in Salmonella

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3], [4] and [5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7], [8], [9] and [10], or as a consequence of evolutionary processes [11], [12], [13], [14], [15] and [16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that chnages in the mutability of specific loci can be influenced by alterations in DNA topology [10] and [17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not ‘directed’ and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.     

FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.     

Evaluation of <Emphasis Type="Italic">Galleria mellonella</Emphasis> larvae as an in vivo model for assessing the relative toxicity of food preservative agents

Larvae of Galleria mellonella are widely used for evaluating the virulence of microbial pathogens and for measuring the efficacy of anti-microbial agents and produce results comparable to those that can be obtained using mammals. In this work, the suitability of using G. mellonella larvae to measure the relative toxicity of a variety of food preservatives was evaluated. The response of larvae to eight commonly used food preservatives (potassium nitrate, potassium nitrite, potassium sorbate, sodium benzoate, sodium nitrate, sodium chloride, sodium nitrite and sodium acetate) administered by feeding or by intra-haemocoel injection was measured. A significant correlation between the LD₅₀ (R ²?=?0.8766, p?=?0.0006) and LD₈₀ (R ²?=?0.7629, p?=?0.0046) values obtained due to oral or intra-haemocoel administration of compounds was established. The response of HEp-2 cells to the food preservatives was determined, and a significant correlation (R ²?=?0.7217, p?=?0.0076) between the LD₅₀ values of the compounds administered by feeding in larvae with the IC₅₀ values of the compounds in HEp-2 cells was established. A strong correlation between the LD₅₀ values of the eight food preservatives in G. mellonella larvae and rats (R ²?=?0.6506, p?=?0.0156) was demonstrated. The results presented here indicate that G. mellonella larvae may be used as a model to evaluate the relative toxicity of food preservatives, and the results show a strong positive correlation to those obtained using established cell culture and mammalian models.     

Improvements in single-use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance

Single-use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO-K1, and CHO-DP12, cultured in media conditioned in an older single-use bioreactor (SUB) film (F-1) and a newer generation film (F-2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F-1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F-2 film. Proteomic profiling of CHO cells cultured in the F-1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F-2 films when compared to F-1 films including bDtBPP, potentially explaining improved CHO cell growth in F-2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess.     

Quantitative detection of C-reactive protein using phosphocholine-labelled enzyme or microspheres

C-reactive protein (CRP) is a positive, acute-phase protein. Plasma levels rise dramatically in response to tissue injury or inflammation and fall rapidly after recovery or treatment. Antibody-based human CRP test systems do not readily detect CRP from other animals due to the species specificity of antibodies directed against human CRP. Thus, generic systems for CRP detection, based solely on the interaction between CRP and phosphocholine (PC), have been developed. PC-bovine serum albumin (PC-BSA) conjugates were produced and either labeled with horseradish peroxidase to facilitate CRP detection in a CRP enzyme-linked sorbent assay (ELSA) or coupled to carboxy-modified microspheres to facilitate the nonenzymatic, turbidimetric detection of CRP. The CRP-ELSA is a competitive assay, where the total assay time is 45 min, the assay sensitivity is 1.06 mg/L CRP, and the dynamic range of the assay is 0-500 mg/L. When PC-BSA conjugate is covalently coupled to carboxylated microspheres, agglutination occurs in the presence of CRP, the extent of which depends on the quantity of CRP present in the sample. Total assay time is 5 min with a dynamic range of 25-500 mg/L. Both assay formats are capable of accurately detecting human CRP and the CRP-ELSA can detect canine CRP as a disease state indicator.     

Cdk5 is essential for synaptic vesicle endocytosis

Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.     

Unmodified calcium concentrations in tumour necrosis factor receptor subtype-mediated apoptotic cell death

Partial bladder outlet obstruction of the rabbit bladder results in a rapid increase in mass characterized by remodeling of the bladder wall.In this study we investigated the effect of partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium, and on blood flow using a fluorescent microsphere technique. Transverse sections of bladder wall were examined after 0 (unobstructed), 1, 3, 5, 7, and 14 days of obstruction. The microvasculature of obstructed rabbit bladder mucosa and detrusor smooth muscle apparently increased relative to augmentation of these compartments, while new vessels appeared in the thickening serosa. These vascular changes correlated with results showing that, at 1 week after obstruction, blood flow (ml/min/g tissue) to the mucosa and detrusor was unchanged.Thickening of the serosa, apparent after 1 day of obstruction, began before its vascularization. Then, 1 week post-obstruction, there was significant microvessel formation in the transition region between the detrusor smooth muscle and the increasing serosa; after 2 weeks, the entire serosa was vascularized. The vascularization of the muscle-serosal transition region and then the remaining serosa apparently precedes fibroblast differentiation, providing blood supply and thus metabolic support for this process.All obstructed rabbit bladders in this study were in a state of compensated function based on their weights. Our working hypothesis is that blood flow per unit tissue mass is normal in compensated obstructed bladders, thus allowing for normal contractile function and cellular metabolism. The results of this study indicate the presence of an augmented microvasculature in compensated obstructed rabbit bladders that provides adequate blood perfusion for normal function.