Hydrolysis of triacetin catalyzed by immobilized lipases: effect of the immobilization protocol and experimental conditions on diacetin yield

The effect of the immobilization protocol and some experimental conditions (pH value and presence of acetonitrile) on the regioselective hydrolysis of triacetin to diacetin catalyzed by lipases has been studied. Lipase B from Candida antarctica (CALB) and lipase from Rhizomucor miehei (RML) were immobilized on Sepabeads (commercial available macroporous acrylic supports) activated with glutaraldehyde (covalent immobilization) or octadecyl groups (adsorption via interfacial activation). All the biocatalysts accumulated diacetin. Covalently immobilized RML was more active towards rac-methyl mandelate than the adsorbed RML. However, this covalent RML preparation presented the lowest activity towards triacetin. For this reason, this preparation was discarded as biocatalyst for this reaction. At pH 7, acyl migration occurred giving a mixture of 1,2 and 1,3 diacetin, but at pH 5.5, only 1,2 diacetin was produced. Yields were improved at acidic pH values and in the presence of 20% acetonitrile (to over 95%). RML immobilized on octadecyl Sepabeads was proposed as optimal preparation, mainly due to its higher specific activity. Each enzyme preparation presented very different properties. Moreover, changes in the reaction conditions affected the various immobilized enzymes in a different way.     

Sampling strategies for square and boll-feeding plant bugs (Hemiptera: Miridae) occurring on cotton

Sampling methods for square and boll-feeding plant bugs (Hemiptera: Miridae) occurring on cotton, Gossypium hirsutum L., were compared with the intent to assess if one approach was viable for two species occurring from early-season squaring to late bloom in 25 fields located along the coastal cotton growing region of south Texas. Cotton fleaphopper, Pseudatomoscelis seriatus (Reuter), damages squares early-season and dominated collections using five sampling methods (approximately 99% of insects collected). A major species composition shift occurred beginning at peak bloom in coastal fields, when verde plant bug, Creontiades signatus Distant, represented 55-65% of collections. Significantly more cotton fleahoppers were captured by experienced samplers with the beat bucket and sweep net than with the other methods (30-100% more). There were more than twice as many verde plant bugs captured by experienced and inexperienced samplers with the beat bucket and sweep net than captured with the KISS and visual methods. Using a beat bucket or sweep net reduced sampling time compared with the visual method for the experienced samplers. For both species, comparing regressions of beat bucket-based counts to counts from the traditional visual method across nine cultivar and water regime combinations resulted in only one combination differing from the rest, suggesting broad applicability and ability to translate established visual-based economic thresholds to beat bucket-based thresholds. In a first look at sample size considerations, 40 plants (four 10-plant samples) per field site was no more variable than variation associated with larger sample sizes. Overall, the beat bucket is much more effective in sampling for cotton fleahopper and verde plant bug than the traditional visual method, it is more suited to cotton fleahopper sampling early-season when plants are small, it transitions well to sample for verde plant bug during bloom, and it performs well under a variety of soil moisture conditions and cultivar selections.     

Effects of leptin replacement on hypothalamic-pituitary growth hormone axis function and circulating ghrelin levels in ob/ob mice

Leptin-deficient obese mice (ob/ob) have decreased circulating growth hormone (GH) and pituitary GH and ghrelin receptor (GHS-R) mRNA levels, whereas hypothalamic GH-releasing hormone (GHRH) and somatostatin (SST) expression do not differ from lean controls. Given the fact that GH is suppressed in diet-induced obesity (a state of hyperleptinemia), it remains to be determined whether the absence of leptin contributes to changes in the GH axis of ob/ob mice. Therefore, to study the impact of leptin replacement on the hypothalamic-pituitary GH axis of ob/ob mice, leptin was infused for 7 days (sc), resulting in circulating leptin levels that were similar to wild-type controls (approximately 1 ng/ml). Leptin treatment reduced food intake, body weight, and circulating insulin while elevating circulating n-octanoyl ghrelin concentrations. Leptin treatment did not alter hypothalamic GHRH, SST, or GHS-R mRNA levels compared with vehicle-treated controls. However, leptin significantly increased pituitary GH and GHRH-R expression and tended to enhance circulating GH levels, but this latter effect did not reach statistical significance. In vitro, leptin (1 ng/ml, 24 h) did not affect pituitary GH, GHRH-R, or GHS-R mRNA but did enhance GH release. The in vivo effects of leptin on circulating hormone and pituitary mRNA levels were not replicated by pair feeding ob/ob mice to match the food intake of leptin-treated mice. However, leptin did prevent the fall in hypothalamic GHRH mRNA and circulating IGF-I levels observed in pair-fed mice. These results demonstrate that leptin replacement has positive effects on multiple levels of GH axis function in ob/ob mice.     

Central B-cell tolerance: where selection begins

The development of an adaptive immune system based on the random generation of antigen receptors requires a stringent selection process that sifts through receptor specificities to remove those reacting with self-antigens. In the B-cell lineage, this selection process is first applied to IgM⁺ immature B cells. By using increasingly sophisticated mouse models, investigators have identified the central tolerance mechanisms that negatively select autoreactive immature B cells and prevent inclusion of their antigen receptors into the peripheral B-cell pool. Additional studies have uncovered mechanisms that promote the differentiation of nonautoreactive immature B cells and their positive selection into the peripheral B-cell population. These mechanisms of central selection are fundamental to the generation of a naïve B-cell repertoire that is largely devoid of self-reactivity while capable of reacting with any foreign insult.B-cell generation in the bone marrow of adult mammals occurs through a tightly controlled developmental process (Fig. 1). Productive rearrangement of immunoglobulin heavy (IgH) and light (IgL) chain gene segments in B lymphocyte precursor cells, in addition to the expression of Ig-α (CD79a) and Ig-β (CD79b), result in the generation and expression on the cell surface of a mature B-cell antigen receptor (BCR). Whereas the combination of Ig H and L chains determines the antigenic specificity of the newly formed BCR, their association with Ig-α and Ig-β allows transduction of a signal inside the cell that directs cell fate. Developing B cells first express a mature BCR on the cell surface in the form of IgM and as such are classified as immature B cells (Fig. 1) (Hardy et al. 1991; Pelanda et al. 1996). It is at the immature B-cell stage that the BCR is tested for the first time for reactivity against autoantigens. This test determines whether the immature B cell and the antibody it expresses on the surface will be selected into the peripheral B-cell repertoire. Central B-cell tolerance, in fact, refers to the process that negatively selects newly generated immature B cells that react with a self-antigen in the bone marrow environment. This is considered the first checkpoint of B-cell tolerance, and the results of this checkpoint are fundamental to the generation of a naïve repertoire that contains foreign reactive antibodies and is largely devoid of self-reactive specificities.Open in a separate windowFigure 1.Schematic representation of B-cell development and Ig loci in mice. Large pro-B cells initiate Ig gene rearrangement at the IgH locus. Expression of a H chain following a productive V_HD_HJ_H recombination event promotes the differentiation of large pre-B cells in which the expression of pre-BCR (H chain pairing with surrogate light chains) results in the clonal expansion of H chain-positive pre-B cells and the development of small pre-B cells. Expression of conventional L chains following productive rearrangements at the IgL chain loci in small pre-B cells promotes the development of a diverse population of IgM⁺ immature B cells, which then differentiate into IgM⁺IgD⁺ transitional B cells. The scheme of mouse Ig H, κ, and λ loci (not to scale) indicate the presence of V (white rectangles), D (black vertical lines), J (brown vertical lines; a dashed line indicates a nonfunctional element), and C (black rectangles; a gray rectangle indicates a nonfunctional element) gene segments. The scheme does not represent the number of V_H, D_H, and Vκ gene segments in the actual Ig loci.On passing this central checkpoint, immature B cells continue to differentiate into transitional and mature B cells before and after they travel to the spleen (Loder et al. 1999; Allman et al. 2001; Su and Rawlings 2002; Tarlinton et al. 2003). Analysis of the bone marrow early immature B-cell repertoire indicates that a staggering 50%–75% of these cells express BCRs that are specific for self-antigens, both in mice and humans (Grandien et al. 1994; Wardemann et al. 2003). Similar studies performed on cell populations at the other end of this central checkpoint, namely, transitional and naïve mature B cells in spleen and blood, show a much lower frequency (20%–40%) of cells expressing autoreactive antibodies (Grandien et al. 1994; Wardemann et al. 2003), demonstrating the stringency and limitation of this initial selection step. Moreover, individuals affected by autoimmune disease such as lupus erythematosus or rheumatoid arthritis bear many more autoreactive cells in their new emigrant and naïve B-cell populations (Samuels et al. 2005; Yurasov et al. 2005), indicating a defect in central (and/or peripheral) B-cell selection. Thus, it seems important that the development of autoreactive immature B cells be constrained to prevent the potential occurrence of autoimmunity. However, there are also reasons to believe that the high frequency of autoreactive specificities generated during primary Ig gene rearrangements may be necessary for the generation of the peripheral B-cell repertoire (Pelanda et al. 1997; Kohler et al. 2008). Indeed, a fraction of autoreactive immature B cells, those manifesting a low level of self-reactivity, do bypass the central checkpoint of tolerance and differentiate into mature B cells (Hayakawa et al. 2003; Wardemann et al. 2003; Wen et al. 2005). The inclusion of these weakly self-reactive B cells in the peripheral B-cell repertoire may allow recognition of a broader spectrum of foreign molecules, potentially decreasing the negative impact of infections, especially at early stages (Mouquet et al. 2010).What are the rules that govern the selection of immature B cells? Most studies of central tolerance have been conducted by following the selection of B cells expressing BCRs displaying well-defined reactivity for natural or synthetic self-antigens. This has been accomplished through the use of Ig transgenic mice in which developing B cells have been altered to carry prerearranged Ig H and L chain genes encoding antibodies of defined antigen specificity and reactivity. Here we review some of these studies, what we have learned from them, and open questions that still await answers.     

Interleukin-6 is a potential biomarker for severe pandemic H1N1 influenza A infection

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.     

Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells

Background  

cDNA microarrays are a powerful means to screen for biologically relevant gene expression changes, but are often limited by their ability to detect small changes accurately due to "noise" from random and systematic errors. While experimental designs and statistical analysis methods have been proposed to reduce these errors, few studies have tested their accuracy and ability to identify small, but biologically important, changes. Here, we have compared two cDNA microarray experimental design methods with northern blot confirmation to reveal changes in gene expression that could contribute to the early antiproliferative effects of neuregulin on MCF10AT human breast epithelial cells.     

Late toxicity for prostate cancer patients treated with hypofractionated helical tomotherapy

AimThe purpose of this study is to evaluate the long term tolerability of hypofractionated helical tomotherapy (HT) in localized prostate cancer patients.BackgroundPrevious hypofractionated schedules with conventional RT were associated with excessive toxicity, likely due to inadequate sophistication of treatment delivery. There are few data about late toxicity after HT.Materials and methodsWe evaluated 38 patients with primary adenocarcinoma of the prostate. There were 9 (24%), 15 (39%), and 14 (37%) patients with high, intermediate, and low risk, respectively. Patients were treated with hypofractionated HT from May 2008 to February 2011. Hypofractionation regimens included: 68.04 Gy at 2.52 Gy/fraction (N = 25; 66%), 70 Gy at 2.5 Gy/fraction (N = 4; 11%) and 70.2 Gy at 2.6 Gy/fraction (N = 9; 23%). Late genitourinary (GU) and gastrointestinal (GI) toxicity was scored using the Radiation Therapy Oncology Group scoring system.ResultsMedian age at diagnosis was 70 years (range 49–80) and median follow-up, 5.8 years. Late grade 1, 2 and 3 GI toxicity were 13%, 24%, and 2.6%, respectively. Late grade 1, 2, 3 GU toxicity were 29%, 21%, and 8%, respectively. Sexual toxicity was evaluated in 19 patients to be grade 1, 2 in 11% and grade 3 in 16%. Multivariate analysis showed that patients with higher values of rectum V50 associated with late GI toxicity (P = 0.025). Patients with PSA ≤8 (P = 0.048) or comorbidities (P = 0.013) at diagnosis were associated with higher late GU toxicity. Additionally, PSA ≤8 also associated with moderate (grade ≥2) late GU toxicity in the multivariate analysis (P = 0.028).ConclusionsHypofractionated HT can be delivered safely with limited rates of moderate and severe late toxicity. The proportion of the rectum that receives a moderate and high dose, having comorbidities, and PSA at diagnosis seem to associate with long term toxicity.     

Nutritional Requirements of <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> in a Chemically Defined Medium

This study was undertaken to determine the nutritional requirements of Lactobacillus delbrueckii subsp. lactis and to develop a minimal chemically defined medium that supports sustained growth of these microorganisms. The single-omission technique was applied to each component of complete chemically defined medium in order to determine the nutritional requirements. L. delbrueckii subsp. lactis was prototrophic for alanine, glycine, aspartic acid, asparagine, glutamine, threonine, and proline. The lysine requirement was strain-dependent. Magnesium was the only essential oligoelement. These microorganisms also required uracil and guanine and adenine as pyrimidine and purine sources, respectively. In view of the nutritional requirements we designed a new minimal defined medium which supports sustained growth of L. delbrueckii subsp. lactis. This medium is simple and well defined, and should be preferable to complex media for conducting future biochemical, physiological, and genetic studies on L. delbrueckii subsp. lactis.     

Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction

Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is major port of entry and exit. The present study evaluated the clinical application of PCR for detection of M. leprae DNA in nasal mucosa biopsies in untreated leprosy patients (52) and their contacts (99) from the State Reference Center in Sanitary Dermatology and Leprosy, Uberlandia, MG, Brazil. PCR detection of a 372-base pair DNA fragment from M. leprae was accomplished in 36 (69.2%) patients, from which 34 (91.9%) of them were multibacillaries. Furthermore, PCR was positive in 3 (16.7%) of 18 slit-skin smear negative, 4 (25.0%) of 16 skin lesion BI negative, 8 (33.3%) of 24 nasal mucosa BI negative patients, and 10 of 99 contacts (10.1%). The presence of bacilli in 10.1% of the contacts may potentially reflect an occult leprosy, and these patients must be accompanied, followed by a chemoprophylaxy treatment. Considering all PCR results against clinical and BI classification of patients and controls, we have found a sensitivity of 69.2%, a specificity of 89.9%, and an accuracy of 82.8%. It has been demonstrated here through PCR of nasal biopsies that the bacillus invades the mucosa, passing through the nasal inferior turbinate to reach peripheral blood. Therefore, the molecular investigation of invasive nasal biopsies by PCR tests has proven to be useful in defining patients of higher risk of transmission and risk-group contacts, which is an important step to reach the World Health Organization objective towards the elimination of leprosy as a public health problem.