Impact of food texture modifications on oral processing behaviour, bolus properties and postprandial glucose responses.

Several studies have demonstrated food texture manipulation on oral processing behaviour (OPB). We explored the effect of texture-differences of equivalent carbohydrate load on OPB, bolus properties and postprandial glycaemic responses (PPG). In a randomised cross-over, within-subjects, non-blinded design, healthy male participants (N = 39) consumed fixed portions of white rice (WR) and rice cake (RC) while being video recorded to measure microstructural eating behaviours. PPG was compared between test foods over a period of 120-min, and the bolus properties and saliva uptake at swallow were measured for both test foods. RC displayed higher instrumental hardness, chewiness and Young's modulus than WR (p = 0.01), and participants perceived RC as more springy and sticky than WR (p < 0.001). The RC meal was chewed more per bite (p < 0.001) and consumed at a faster eating rate (p = 0.033) than WR. WR bolus particles were smaller at swallow (p < 0.001) with a larger total surface area (p < 0.001), compared to RC. The glucose response for RC was significantly higher during the first 30-min postprandial period (p = 0.010), and lower in the later (30-120 min) postprandial period (p = 0.031) compared to WR. Total blood glucose iAUC did not differ significantly between WR and RC meals despite their large differences in texture, OPB and bolus properties. Oro-sensory exposure time was a significant predictor of glucose iAUC30min for both test meals (RC, p = 0.003; WR, p = 0.029). Saliva uptake in the bolus was significantly positively associated with blood glucose during the first 30-min postprandial period for the RC meal (p = 0.008), but not for WR. We conclude that food texture modifications can influence OPB and bolus properties which are key contributors to the dynamic evolution of the glycaemic response. Total blood glucose responses were the same for both test foods, though differences in oral processing and bolus properties influenced temporal changes in PPG.

Sweetness but not sourness enhancement increases acceptance of cucumber and green capsicum purees in children.

For children it is important to consume enough vegetables to establish healthy dietary patterns. Taste acceptance is an important factor contributing to food choice and consumption. Sweetness and sourness enhancement can increase acceptance of specific foods in children. The aim of this study was to determine the effect of sweetness and sourness enhancement on acceptance of cucumber and green capsicum purees in 5-6-year-old children. Three concentrations of sucrose (2, 5 and 10%) and citric acid (0.05, 0.08 and 0.15%) were added to cucumber and green capsicum purees. Children (n = 70, 5.7 ±â€¯0.5 yrs) assessed acceptance of the vegetable purees using a 5-point hedonic facial scale. Sweetness enhancement significantly increased acceptance of cucumber purees (5 and 10% sucrose) and green capsicum purees (2 and 10% sucrose) compared to unmodified purees. Sourness enhancement (0.05, 0.08 and 0.15% citric acid) did not significantly influence acceptance of cucumber and green capsicum purees compared to unmodified purees. Children differed in acceptance of vegetable purees with added sucrose and citric acid. Sweetness likers (cucumber 77.1%, green capsicum 58.6%) accepted sucrose concentrations better than sweetness non-likers in both vegetables. Sourness likers (cucumber 50.0%, green capsicum 44.3%) accepted medium and high concentrations of citric acid better than sourness non-likers in cucumber and all citric acid concentrations in green capsicum. We conclude that enhancement of sweetness increases acceptance of cucumber and green capsicum purees in most children whereas enhancement of sourness is better accepted by only a few children. This study highlights the challenge to get children to better accept vegetables, since only sweetness enhancement improved acceptance while addition of sucrose is undesirable. For a small subset of children enhancing sourness might be an alternative strategy to increase acceptance of vegetables.

Effect of outer water phase composition on oil droplet size and yield of (w1/o/w2) double emulsions.

In this study the effect of various emulsifiers (whey protein isolate (WPI), Na-caseinate, and Tween 20) and thickeners (xanthan and pectin) present in the outer water phase, w2, on oil droplet size and yield of the inner water phase, w1, of (w1/o/w2) double emulsions was investigated. Double emulsions stabilized by Tween 20 had smaller oil droplet sizes and higher yields in comparison to emulsions stabilized by WPI and Na-caseinate. Gelation of the inner water droplets w1 increased yield by 20% for all emulsifiers. Upon the addition of thickeners, the increasing viscosity of the outer water phase, w2, facilitated oil droplet breakup. This resulted in smaller oil droplets and lower yields. When pectin was used as a thickener, in comparison to xanthan, an additional decrease in yield was observed. The yield decreased to values close to zero indicating that all inner water droplets w1 were lost during emulsification. We conclude that type of hydrophilic emulsifier, properties of inner water droplets, viscosity ratio of continuous and dispersed phase, as well as type of thickener influence oil droplet size and yield of w1 phase of double emulsions. This work provides a better understanding of how composition influences the properties of double emulsions and how this can be used to design double emulsions as fat replacers in more complex food systems.

The role of smell, taste, flavour and texture cues in the identification of vegetables.

It has been shown that the identification of many foods including vegetables based on flavour cues is often difficult. The effect of providing texture cues in addition to flavour cues on the identification of foods and the effect of providing taste cues only on the identification of foods have not been studied. The aim of this study was to assess the role of smell, taste, flavour and texture cues in the identification of ten vegetables commonly consumed in The Netherlands (broccoli, cauliflower, French bean, leek, bell pepper, carrot, cucumber, iceberg lettuce, onion and tomato). Subjects (n = 194) were randomly assigned to one of four test conditions which differed in the sensory cues available for vegetable identification: taste, smell (orthonasal), flavour (taste and smell) and flavour-texture (taste, smell and texture). Blindfolded subjects were asked to identify the vegetable from a list of 24 vegetables. Identification was the highest in the flavour-texture condition (87.5%). Identification was significantly lower in the flavour condition (62.8%). Identification was the lowest when only taste cues (38.3%) or only smell cues (39.4%) were provided. For four raw vegetables (carrot, cucumber, onion and tomato) providing texture cues in addition to flavour cues did not significantly change identification suggesting that flavour cues were sufficient to identify these vegetables. Identification frequency increased for all vegetables when perceived intensity of the smell, taste or flavour cue increased. We conclude that providing flavour cues (taste and smell) increases identification compared to only taste or only smell cues, combined flavour and texture cues are needed for the identification of many vegetables commonly consumed in The Netherlands.

Comparison of oro-sensory exposure duration and intensity manipulations on satiation.

Oro-sensory exposure (OSE) is an important factor in the regulation of food intake with increasing OSE leading to lower food intake. Oral processing time and taste intensity both play an important role in OSE but their individual contribution to satiation is unknown. We aimed to determine the independent and combined effects of oral processing time and taste intensity on satiation. Fifty eight participants (23±9y, BMI 22±2kg/m2) participated in a 2×2 factorial randomized crossover study in which they consumed one of four gel-based model foods until satiation during four sessions. Model foods were offered ad libitum and differed in texture (soft or hard texture, yielding shorter and longer oral processing time) and sweetness (low or high intensity). Model foods were isocaloric and were matched for flavor and palatability. Outcome measures were intake of the model food and the microstructure of eating behavior, such as number of chews and eating rate. There was an overall significant effect of texture (p<0.001) but not sweetness (p=0.33) on intake with a 29.2% higher intake of the soft model foods compared to the hard model foods. After correction for palatability the difference in intake between the soft and hard model foods was 21.5% (p<0.001). The number of chews was significantly lower for the soft (10.1±6.2) than for the hard (26.9±6.2) model foods (p<0.001), which resulted in a significantly lower eating rate (soft, 26.3±10.2 and hard, 15.3±7.1g/min, p<0.001). These results show that increasing texture hardness of gel model foods decreases food intake independent of sweet taste intensity. The higher number of chews and faster eating rate may cause this effect. In conclusion, oro-sensory exposure duration rather than taste intensity appears to be the main determinant of food intake.

Influence of double (w1/o/w2) emulsion composition on lubrication properties.

Double (w1/o/w2) emulsions are potential fat replacers in foods. Fats are known for their lubricating properties, which contribute to texture perception. It is therefore of interest to understand how the composition of double emulsions influences lubrication properties. This study focuses on the understanding of the influence of the fraction of inner dispersed aqueous phase w1 and the gelation of the w1 droplets on the lubrication properties of double emulsions. The addition of an inner water phase w1 to the oil droplets decreased friction at low entrainment speeds due to adsorption of the lipophilic emulsifier polyglycerol polyricinoleate (PGPR) at the hydrophobic tribo-surface. At higher entrainment speeds, double emulsions with w1 fractions of up to 6% (corresponding to fat reduction of 20%) displayed comparable tribological behavior as full-fat single (o/w2) emulsions. For double emulsions with gelled w1 droplets at higher w1 fractions of up to 15% (corresponding to fat reduction of 50%), an increase in friction was observed compared to full-fat single (o/w2) emulsions. The increase in friction is probably related to the presence of gelled droplets expelled from the inner w1 into the outer w2 phase, and to the deformability of (w1/o) droplets. Lubrication decreased when gelled particles were expelled from the inner w1 phase to the outer w2 phase. Lubrication also decreased when the deformability of (w1/o) droplets decreased, since less deformable (w1/o) droplets spread less easily on the tribo-pair surface. Knowledge about lubrication properties of double emulsions can be used in future studies to relate composition to sensory perception and develop double emulsions further as fat replacers.

Taste intensities of ten vegetables commonly consumed in the Netherlands.

Bitterness has been suggested to be the main reason for the limited palatability of several vegetables. Vegetable acceptance has been associated with preparation method. However, the taste intensity of a variety of vegetables prepared by different methods has not been studied yet. The objective of this study is to assess the intensity of the five basic tastes and fattiness of ten vegetables commonly consumed in the Netherlands prepared by different methods using the modified Spectrum method. Intensities of sweetness, sourness, bitterness, umami, saltiness and fattiness were assessed for ten vegetables (cauliflower, broccoli, leek, carrot, onion, red bell pepper, French beans, tomato, cucumber and iceberg lettuce) by a panel (n=9) trained in a modified Spectrum method. Each vegetable was assessed prepared by different methods (raw, cooked, mashed and as a cold pressed juice). Spectrum based reference solutions were available with fixed reference points at 13.3mm (R1), 33.3mm (R2) and 66.7mm (R3) for each taste modality on a 100mm line scale. For saltiness, R1 and R3 differed (16.7mm and 56.7mm). Mean intensities of all taste modalities and fattiness for all vegetables were mostly below R1 (13.3mm). Significant differences (p<0.05) within vegetables between preparation methods were found. Sweetness was the most intensive taste, followed by sourness, bitterness, fattiness, umami and saltiness. In conclusion, all ten vegetables prepared by different methods showed low mean intensities of all taste modalities and fattiness. Preparation method affected taste and fattiness intensity and the effect differed by vegetable type.

Descriptive sensory profiling of double emulsions with gelled and non-gelled inner water phase.

The use of double emulsions (w1/o/w2) has been acknowledged as a promising strategy to reduce oil content in several food applications. Despite the potential of double emulsions for oil reduction, their sensory properties have not been investigated. In this study, we investigated sensory perception of double emulsions by descriptive sensory profiling using a trained panel (n=11). Two sets of emulsions with either 30 or 50% dispersed phase fraction were studied. Each set differed in composition (gelled and non-gelled inner w1 phase, gelatin as gelling agent) and fat reduction level (30 to 50%), but was similar in oil droplet size and viscosity. Fat reduction level depended on the amount of water droplets entrapped inside the oil droplets. Emulsions were evaluated on nine attributes describing taste (T), mouth-feel (MF) and after-feel (AF) perception, including thickness (MF), creaminess (MF, AF), fattiness (MF, AF), and cohesiveness (MF). The replacement of oil by small water droplets w1 did not decrease the intensity of fat-related attributes. When inner w1 droplets were gelled, 47wt.% of oil could be replaced while increasing the intensity of fat-related attributes. This indicates that the sensory perception of single and double emulsions with gelled and non-gelled w1 phase is mainly determined by the total oil droplet surface area. The composition of the inner water phase (gelled or not) also influences the sensory perception of double emulsions. We conclude that fat reduction up to 47wt.% can be achieved in double emulsions while maintaining or enhancing fat-related sensory perception.

Lifelong physical exercise delays age-associated skeletal muscle decline.

Aging is usually accompanied by a significant reduction in muscle mass and force. To determine the relative contribution of inactivity and aging per se to this decay, we compared muscle function and structure in (a) male participants belonging to a group of well-trained seniors (average of 70 years) who exercised regularly in their previous 30 years and (b) age-matched healthy sedentary seniors with (c) active young men (average of 27 years). The results collected show that relative to their sedentary cohorts, muscle from senior sportsmen have: (a) greater maximal isometric force and function, (b) better preserved fiber morphology and ultrastructure of intracellular organelles involved in Ca(2+) handling and ATP production, (c) preserved muscle fibers size resulting from fiber rescue by reinnervation, and (d) lowered expression of genes related to autophagy and reactive oxygen species detoxification. All together, our results indicate that: (a) skeletal muscle of senior sportsmen is actually more similar to that of adults than to that of age-matched sedentaries and (b) signaling pathways controlling muscle mass and metabolism are differently modulated in senior sportsmen to guarantee maintenance of skeletal muscle structure, function, bioenergetic characteristics, and phenotype. Thus, regular physical activity is a good strategy to attenuate age-related general decay of muscle structure and function (ClinicalTrials.gov: NCT01679977).

Efficacy of topical tacrolimus for oral lichen planus: real-life experience in a retrospective cohort of patients with a review of the literature.

BACKGROUND AND OBJECTIVE: Management of oral lichen planus (OLP) is challenging and therapeutic options are limited. The use of topical tacrolimus has shown promising results. We reviewed our daily life experience with topical tacrolimus in OLP patients. METHODS: This retrospective unicentre study included all 21 patients with OLP, which were evaluated over a 53-month period and treated with topical tacrolimus. Patients were initially given a topical preparation of 0.1% tacrolimus twice daily. The response to treatment was assessed using a 4-point scale at month 2 and 6: complete response of affected area (CR), major remission (>50%, MR), partial remission (25-50%, PR) and either no response (<25%) or worsening. The pain score was also assessed using a 3-point scale. RESULTS: Four of 21 patients (19%) showed a CR at month 2, whereas at month 6, 7 (33%) had a CR. For patients who reported MR (n = 2) and PR (n = 8) at month 2, the therapy was continued. Of those, at 6 months, three patients showed a CR, while four maintained a PR. The pain score improved during treatment. After 2 months of therapy, eight of 10 patients with an initial high pain score achieved a significant improvement. In patients starting with moderate pain an improvement was observed in one of seven patients. Overall, for three patients there was a complete loss of pain, while in nine there was a reduction. Except for transitory burning sensation and altered taste sensation, no relevant side-effects were reported. CONCLUSION: This retrospective analysis confirms that topical tacrolimus is a valuable therapeutic option in severe or treatment-resistant OLP. Our findings in daily practice suggested nevertheless that the efficacy of topical tacrolimus is overestimated with regard to both complete response and pain reduction.

Physical and sensory characterizations of oral coatings of oil/water emulsions.

The physical and sensory properties of oil coatings on the tongue formed by five oil/water emulsions varying in oil content were investigated. A total of 20 subjects processed orally each emulsion for 30 s in triplicate. In vivo fluorescence measurements at the front and back of the anterior tongue were made to quantify the oil fraction deposited at different time points. Calibration lines relating fluorescence intensity to oil fraction were determined using pig tongues at 37.5 °C to mimic oral conditions. The oil fraction on the tongue increased linearly with an increasing oil content of the emulsions. The oil fraction deposited at the back of the anterior tongue was 1.5-2.0× larger than at the front. The intensity of sensory attributes describing after-feel perception was related to the oil fraction by Weber-Fechner's law. This study uses in vivo fluorescence to study food behavior in the mouth and unravel new insights in after-feel perception of emulsions.

[Extracorporeal shock wave therapy as a treatment of a non-healing chronic leg ulcer]. / Extrakorporale Stoßwellentherapie eines komplizierten chronischen Ulcus cruris venosum.

Extracorporeal shock waves are defined as a sequence of sonic pulses characterized by high peak pressure over 100 MPa, fast pressure rise, and short lifecycle. In the 1980s extracorporeal shock wave lithotripsy (ESWL) was first used for the treatment of urolithiasis. Orthopedic surgeons use extracorporeal shock wave therapy (ESWT) to treat non-union fractures, tendinopathies and osteonecrosis. The first application of ESWT in dermatology was for recalcitrant skin ulcers. Several studies in the last 10 years have shown that ESWT promotes angiogenesis, increases perfusion in ischemic tissues, decreases inflammation, enhances cell differentiation and accelerates wound healing. We successfully treated a non-healing chronic venous leg ulcer with ESWT. Furthermore we observed an improvement of the lymphatic drainage after application of ESWT. We are confident that ESWT is a non-invasive, practical, safe and efficient physical treatment modality for recalcitrant leg ulcers.

A systematic review on the effectiveness of medical training therapy for subacute and chronic low back pain.

Medical training therapy (MTT) to improve muscular strength and endurance follows evidence based guidelines and is increasingly recommended to patients suffering from subacute and chronic back pain (LBP). This study investigated whether MTT was effective in reducing pain and improving function in patients with subacute or chronic LBP. Data sources were MEDLINE, EMBASE, CINAHL, Pedro, Cochrane Central Register of Controlled Trials. We included RCTs that examined exercise or MTT in adult patients with LBP compared to placebo, no intervention or other interventions. Study outcomes had to include at least one of the following: pain intensity; functional status, absenteeism. Two independent reviewers performed quality assessment. Visual analogue scale ratings ranging from 0-10 MTT quantified the MTT aspects of the intervention. Studies with rating scores >7.5 were included. We identified only 2 studies that examined the effectiveness of MTT. Both trials, one was of high quality, found MTT to decrease pain and improve function significantly better than therapy of uncertain effectiveness. There is moderate evidence that would support the effectiveness of MTT in the treatment chronic LBP. Future high quality RCT will have to clarify whether MTT is effective and would be superior to other forms of therapeutic exercise.

Effect of NaCl and sucrose tastants on protein composition of oral fluid analysed by SELDI-TOF-MS.

During eating, human saliva is secreted into the oral cavity by salivary glands. The relative contribution of different glands to total salivary flow rate depends, among other factors, on the tastants in the food. Few reports indicated that also the salivary protein composition depends on the tastant make-up of the food. We studied the influence of sodium-chloride- and sucrose solutions on the presence of proteins in the M(r) range 2-20kDa in whole saliva. Upon oral stimulation with a sodium chloride solution, a sucrose solution or water, we collected whole saliva from 14 volunteers after t=1 min, t=11 min and t=20 min. Saliva protein profiles were analysed by SELDI-TOF-MS. SELDI-TOF-MS intensities of m/z values representing different protein masses were compared between subjects, tastants and time conditions. For subsets of the 33 detected masses, significant effects were observed for all factors, with most masses involved in the Subjects effect: m/z(Subjects)>m/z(Time×Stimulus)>m/z(Stimulus)>m/z(Time). Most effects on saliva protein composition were observed at t=1 min, whilst almost no effects were observed at t=11 min and t=20 min. When considering the Stimulus×Time interaction, we identified four different stimulus-response patterns. Proteins identified in the present study, and attributed to specific glands or tissues in literature, were used to associate stimulus-response patterns with tissue provenances. Observed stimulus-response patterns were not uniquely associated to particular glands and tissues. Hence, there was no evidence of the involvement of particular tissues or glands in tastant-specific protein responses. In conclusion, oral stimulation with different tastants affects salivary protein composition in a protein- and stimuli dependent way, which seems not be associated with any specific tissues or glands of origin.

Effects of 8 weeks of vibration training at different frequencies (1 or 15 Hz) in senior sportsmen on torque and force development and of 1 year of training on muscle fibers.

OBJECTIVE: To examine the effects of 8 weeks of vibration training at different frequencies (1 and 15 Hz) on maximal isometric torque and force development in senior sportsmen, and of 1 year of heavy-resistance and vibration trainings on muscle fibers. METHODS: Seven healthy senior sportsmen (mean age: 69.0 +/- 5.4 years) performed an 8 weeks of strength training of knee extensors. Vibrations were applied vertically to the axis of movement during training. One leg of each subject was trained at a frequency of 1 Hz, while the other leg was trained at 15 Hz. Measures of isometric peak torque (at knee-angles of 60, 90 and 120 degrees ) and force development were recorded before and after training. Four sportsmen continued a year-long heavy-resistance training adding every second week a session of vibration training. After training, muscle biopsies were harvested from their quadriceps muscles and used for structural analyses. Morphometry of muscle fibers was performed by light microscopy. Immunohistochemistry using anti-MHCemb and anti-N-CAM antibodies was performed to measure potential muscle damage. Data from muscle morphometry were compared to that of a series of vastus lateralis biopsies harvested from 12 young sportsmen and four healthy elderly. RESULTS: Our results showed a significant increase in isometric peak torque at both 1 and 15 Hz vibration frequency in all three measured angles of the knee. There was no significant difference between the two frequencies, but we could find a higher increase in percentage of maximum power after the 1 Hz training. The results of force development showed a slight increase at the 1 Hz training in measured time frames from 0 to 50 and 200 ms, without statistical significance. A trend to significance was found at the 1 Hz training at the time window up to 200 ms. The 15 Hz training showed no significant changes of force development. Muscle biopsies show that the muscles of these well trained senior sportsmen contain muscle fibers which are 35% larger than those of sedentary elderly and, unexpectedly, 10% larger than those of young sportsmen. Despite 1 year of heavy resistance and vibration training, no evidence of muscle damage or denervation/reinnervation could be observed by light microscopy analyses, ATPase histochemistry and immunohistochemistry using anti-N-CAM or anti-MHC-emb antibodies. DISCUSSION: Integration of vibration to conventional strength training in elderly sportsmen induces similar improvement of isometric peak torque and force development independently from the vibration frequency after 8 weeks of training, and long-term results in the surprising evidence of hypertrophic muscle fibers larger than those of young active sportsmen. The observation that the vibration training with low frequency is safe opens the possibility to test these rehabilitation procedures in sedentary elderly.

Serum levels of tumour necrosis factor-alpha and interleukin-6 and their correlation with body mass index, weight loss, appetite and survival rate--preliminary data of Viennese outpatients with metastatic cancer during palliative chemotherapy.

The serum cytokine levels (in particular interleukine-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha)) of 61 advanced stage cancer patients receiving palliative chemotherapy as outpatients were determined with quantikine immunoassays. The values were correlated with body mass index (BMI), weight loss and appetite. Furthermore cytokine levels of patients who have died within one year were compared with those of patients who have survived more than a year. Serum levels of IL-6 (median: 1.93 pg/ml, range: 0.32-42.87) and of TNF-alpha (median: 2.55 pg/ml, range: 1.03-34.06) did not correlate with BMI, weight loss and appetite. Serum IL-6 levels of patients with survival time less than one year were significantly higher than the levels of patients who survived more than one year, no significant differences in TNF-alpha serum levels were evident. The data of this observation are consistent with current literature. Due to changes in serum levels of proinflammatory cytokines in response to chemotherapy and additional therapy, it is unlikely that IL-6 and TNF-alpha can be used as independent indicators for weight loss and appetite. Nevertheless, high serum levels of IL-6 correlate with short-time mortality.

Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae.

In bacteria, adaptive responses to environmental stimuli are often initiated by two-component signal transduction systems (TCS). The prototypical TCS comprises two proteins: a histidine kinase (HK, hk) and a response regulator (RR rr). Recent research has suggested that compounds that inhibit two-component systems might have good antibacterial activity. In order to identify TCS that are crucial for growth or virulence of Streptococcus pneumoniae, we have examined the genomic sequence of a virulent S. pneumoniae strain for genes that are related to known histidine kinases or response regulators. Altogether 13 histidine kinases and 13 response regulators have been identified. The protein sequences encoded by these genes were compared with sequences deposited in public databases. This analysis revealed that two of the 13 pneumococcal TCSs have been described before (ciaRH and comDE) and two are homologous to the yycFG and the phoRP genes of Bacillus subtilis. All the pneumococcal response regulators contain putative DNA binding motifs within the C-terminal output domain, implying that they are involved in transcriptional control. Two of these response regulators are obviously the first representatives of a new subfamily containing an AraC-type DNA-binding effector domain. To assess the regulatory role of these transcription factors, we disrupted each of the 13 response regulator genes by insertional mutagenesis. All the viable mutant strains with disrupted response regulator genes were further characterized with regard to growth in vitro, competence, and experimental virulence. Two response regulator genes could not be inactivated, indicating that they may regulate essential cellular functions. The possibility of using these systems as targets for the development of novel antibacterials will be discussed.

Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus.

The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow single well defined crystals in the microbatch system which belonged to the space group C2 and diffracted isotropically to approximately 2 A resolution.

Integrational plasmids for the tetracycline-regulated expression of genes in Streptococcus pneumoniae.

Plasmids for the tetracycline regulated gene expression in Streptococcus pneumoniae have been developed. The plasmids were used for the tetracycline-dependent production of firefly luciferase in streptococci. The production of luciferase can be induced fivefold by the addition of tetracycline. By using two promoters of different strength and depending on the presence or absence of tetracycline, an 80-fold range of luciferase activities can be covered. The system was also used to construct strains that depend on the addition of tetracycline for the production of the A subunit of DNA gyrase, an essential streptococcal protein. The growth of such a strain depends on the addition of tetracycline to the medium. In the absence of tetracycline, the cells cease to grow and are not viable. The system presented in this report should be useful for the characterization of gene networks in S. pneumoniae. It especially allows one to study the function of essential genes that can not be investigated by standard knock-out techniques.

Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.