Protection of DNA by alpha/beta-type small,acid-soluble proteins from Bacillus subtilis spores against cytosine deamination

Spores of Bacillus subtilis contain high levels of proteins, termed alpha/beta-type small, acid-soluble proteins (SASP), that protect the spore's DNA against different types of DNA damage. We tested one such protein, SspC, and two of its variants for their ability to protect plasmid DNA against hydrolytic deamination of cytosine to uracil. If unrepaired, such damage to DNA causes C to T mutations. We found that one SspC variant, SspC(Delta 11-D13K), protected DNA against cytosine deamination at two different temperatures (45 and 70 degrees C) and pH values (5.2 and 7.9), reducing the rate of deamination by as much as 10-fold. At 70 degrees C, pH 7.9, the wild-type SspC and its variant, SspC(Delta 11), provided little protection against deamination but were effective in protecting DNA at 45 degrees C, pH 7.9. Parallel studies of the abilities of these proteins to protect DNA against restriction digestion revealed that there was a good correlation between the abilities of the proteins to protect against restriction endonucleases and reductions in cytosine deaminations. These results show that the binding of SspC variants to DNA can prevent attack on DNA bases by water and suggest a new general mechanism by which DNA-binding proteins in cells may be able to protect chromosomes from endogenous and exogenous reactive chemicals by excluding them from the vicinity of DNA.     

Evaluation of the effect of cleaning regimes on biofilms of thermophilic bacilli on stainless steel

AIMS: To determine the mechanism for both the removal and inactivation of 18-h biofilms of a thermophilic Bacillus species that optimally grows at 55 degrees C on stainless steel. METHODS AND RESULTS: The cleaning strategies tested were based on biofilm biochemistry and physiology, and focused on the chemistry of the cleaners, the duration and temperature of the cleaning process and a combination of various cleaners. The success of the cleaning regimes was determined based on the removal of cells and organic debris and the elimination of viable cells. The results confirmed that a caustic (75 degrees C for 30 min) and acid (75 degrees C for 30 min) wash, relied upon heavily in most food processing industries for cleaning-in-place systems, was successful in removing these biofilms. However, any changes in the concentrations of these cleaners or the temperature of cleaning drastically affected the overall outcome. Alternative cleaning agents based on enzymatic or nonenzymatic breakdown of cellular proteins or polysaccharides, surfactant action, use of oxidative attack and free radicals varied in degrees of their success. Combining proteolytic action with surfactants increased wetability and therefore enhanced the cleaning efficiency. CONCLUSIONS: Several procedures, including caustic/acid and enzyme based cleaners, will be satisfactory, provided that the correct process parameters are observed i.e. concentration, time, temperature and kinetic energy (flow). Confirmation of these results should be carried out in a pilot plant through several use/clean cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: Confidence in standard and alternative cleaning procedures for food manufacturing plant to prevent contamination with thermophilic bacilli that threaten product quality.     

Compatibility of osmolytes with Gibbs energy of stabilization of proteins

This study led to the conclusion that naturally occurring osmolytes which are known to protect proteins against denaturing stresses, do not perturb the Gibbs energy of stabilization of proteins at 25 degrees C (DeltaG(D) degrees ) which has been shown to control the in vivo rate of degradative protein turnover (Pace et al., Acta Biol. Med. Germ 40 (1981) 1385-1392). This conclusion has been reached from our studies of heat-induced denaturation of lysozyme, ribonuclease A, cytochrome c and myoglobin in the presence of different concentrations of osmolytes, namely, glycine, proline, sarcosine and glycine-betaine. At a fixed concentration of osmolyte a heat-induced denaturation curve measured by following changes in the molar absorption coefficient of the protein, was analyzed for T(m), the midpoint of the denaturation and DeltaH(m), the enthalpy change of denaturation at T(m). Values of DeltaG(D) degrees were determined with Gibbs-Helmoltz equation using known values of T(m), DeltaH(m) and DeltaC(p), the constant-pressure heat capacity change. It has been observed that T(m) increases with the osmolyte concentration, whereas DeltaG(D) degrees remains unaffected in the presence of the osmolyte. This observation on DeltaG(D) degrees in the presence of osmolytes has been considered in the physiological context.     

Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

Background

Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters.

Methodology/Principal Findings

The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56^th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the ¹H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster’s three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth.

Conclusions and Significance

This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.     

Testing the paradigm that the denaturing effect of urea on protein stability is offset by methylamines at the physiological concentration ratio of 2:1 (urea:methylamines)

The intra- and extracellular urea concentration in many organisms is sufficiently high to destabilize (inhibit) many proteins, yet organisms survive and function. The generally accepted explanation is the counteracting hypothesis, which holds that methylamines stabilize proteins and oppose the deleterious effect of urea. The two osmolytes are typically found at 2:1 concentration ratio (urea:methylamine) under physiological conditions. Does this mean that this ratio holds for all proteins in a cell? The present study tests the counteracting hypothesis by determining the effects of urea and methylamines (trimethylamine N-oxide and sarcosine), singly and in combination at a concentration ratio of 2:1 (urea:methylamine) on the thermal denaturation equilibrium, native state<-->denatured state of three different proteins (alpha-lactalbumin, lysozyme and Ribonuclease-A). We show here that the molar concentration of a methylamine required to offset the denaturing effect of urea at a given concentration is different for different proteins.     

Increased activity of ATP-sulfurylase and increased contents of cysteine and glutathione reduce high cadmium-induced oxidative stress in mustard cultivar with high photosynthetic potential

Cadmium is known to reduce photosynthesis and overall growth of plants. Plants adopt several mechanisms of Cd detoxification, such as accumulation of sulfur-rich compounds, like glutathione (GSH) and its precursor cysteine. The accumulation of GSH is regulated by the activity of ATP-sulfurylase, a rate-limiting enzyme in sulfur assimilation. The carbon of Cys is provided through photosynthesis. Thus, a plant with the higher photosynthetic potential and ATP-sulfurylase activity may have the higher contents of Cys and GSH and therefore may provide for a greater tolerance to Cd stress. Mustard (Brassica juncea L. Czern and Coss.) cvs. Varuna (high photosynthetic potential) and RH30 (low photosynthetic potential) were subjected to 0 and 200 mg Cd/kg soil, and the activity of ATP-sulfurylase, the contents of Cys and GSH, oxidative stress, and activities of antioxidant enzymes were studied. Under 200 mg Cd/kg soil, cv. Varuna showed an increased ATP-sulfurylase activity, the higher contents of Cys and GSH, and the net photosynthetic rate than cv. RH30. In contrast, the activity of superoxide dismutase, the contents of thiobarbituric acid-reactive substances, and H₂O₂, and electrolyte leakage were found to be greater in cv. RH30 showing an increased oxidative stress than cv. Varuna. However, the activities of ascorbate peroxidase and glutathione reductase were greater in cv. Varuna than cv. RH30. The results show that a greater ATP-sulfurylase activity, an enhanced production of Cys and GSH, and an efficient antioxidant enzyme system in the high photosynthetic mustard cv. Varuna helped to the reduce the oxidative stress maintaing high photosynthesis.     

Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli

We have cloned, overexpressed, and purified the catalytic domain (residues Gly106 to Asn268) of human macrophage metalloelastase (MMP-12) in Escherichia coli. This construct represents a truncated form of the enzyme, lacking the N-terminal propeptide domain and the C-terminal hemopexin-like domain. The overexpressed protein was localized exclusively to insoluble inclusion bodies, in which it was present as both an intact form and an N-terminally truncated form. Inclusion bodies were solubilized in an 8 M guanidine-HCl buffer and purified by gel filtration chromatography under denaturing conditions. Partial refolding of the protein by dialysis into a 3 M urea buffer caused selective degradation of the truncated form of the protein, while the intact catalytic domain was unaffected by proteolysis. An SP-Sepharose chromatography step purified the protein to homogeneity and served also to complete the refolding. The purified protein was homogeneous by mass spectrometry and had an activity similar to that of the recombinant enzyme purified from mammalian cells. The protein was both soluble and monodisperse at a concentration of 9 mg/ml. This purification procedure enables the production of 23 mg of protein per liter of E. coli culture and is amenable to large-scale protein production for structural studies.