Isolation and characterization of two closely related beta-carbonic anhydrases of Chlamydomonas reinhardtii

Aquatic photosynthetic organisms such as the green alga, C. reinhardtii respond to low carbon dioxide conditions by inducing a carbon dioxide concentrating mechanism (CCM). Important components of the CCM are carbonic anhydrases (CAs), zinc metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. C. reinhardtii now has nine carbonic anhydrases, three alpha-CAs and six beta-CAs. This dissertation describes the identification and characterization of two closely related beta-CAs, CAH7 and CAH8. These CAs are 63% identical. CAH7 and CAH8 encode proteins of 399 and 333 amino acids, respectively. Both of these CAs are constitutively expressed at the transcript and protein level. Preliminary results of immunolocalization studies localized CAH7 in the chloroplast while CAH8 was localized in the periplasm. Both the CAH7 and CAH8 open reading frames (ORF) were cloned in the overexpression vector pMal-c2x and expressed as recombinant proteins. Activity assay demonstrated that CAH7 and CAH8 are active carbonic anhydrases. The proposed roles for CAH7 and CAH8 are discussed. Previously, insertional mutants were generated to be able to isolate bicarbonate-transporters and other proteins that might be essential for a functional CCM. One of the generated insertional mutants is slc211, a mutant that requires high carnon dioxide for optimum growth. The mutant slc211 had an insertion in the novel gene designated as CIA7. RNA interference was successfully used to reduce the expression of CIA7. The resultant transformants had a growth phenotype similar to slc211 requiring high carbon dioxide for optimum growth. These results suggest that CIA7 is a gene that facilitates growth in C. reinhardtii under low carbon dioxide conditions. The possible functions of CIA7 are discussed

Do cell phones, iPods/MP3 players, siblings and friends matter? Predictors of child body mass in a U.S. Southern Border City Middle School

Objective: This study examines the association of children\u27s (i) micro-social environment, specifically siblings [kin-friends] and friends from school and neighborhood [non-kin-friends], and (ii) ownership of information and communication technologies (ICT), specifically cell phones and iPod/MP3 players, with body mass index percentile (BMIp). Subjects: Fifty-five randomly selected 6th graders with a mean age of 12 years, stratified by gender (23 boys and 32 girls), from a Texas middle school located in a city along the U.S. southern border. Methods: The linear regression of BMIp on number of siblings and of non-kin-friends, and ownership of cell phone and of iPod/MP3 player was examined using two models: M1 was based on the manual selection of predictors from a pool of potential predictors. M2 was derived from the predictors specified in M1 using backward elimination technique. Because sample size was small, the significance of regression coefficients was evaluated using robust standard errors to calculate t-values. Data for predictors were obtained through a survey. Height and weight were obtained through actual anthropometric measurements. BMIp was calculated using the on-line BMI calculator of the Center for Disease Control and Prevention. Results: Findings reveal that children\u27s social environment and ICT ownership predict BMIp; specifically, number of siblings (M2: β = -0.34, p-value \u3c.001), and ownership of iPod/MP3 players (M2: β = 0.33, p-value \u3c.001). These results underscore the importance of family in configuring, and of new personal technical devices (that encourage solitary, and oftentimes sedentary, activities) in predicting child body mass. © 2011 Published by Elsevier Ltd

Comparison of the effects of nitrogen-, sulfur- and combined nitrogen- and sulfur-deprivations on cell growth, lipid bodies and gene expressions in Chlamydomonas reinhardtii cc5373-sta6

Abstract Background Biofuel research that aims to optimize growth conditions in microalgae is critically important. Chlamydomonas reinhardtii is a green microalga that offers advantages for biofuel production research. This study compares the effects of nitrogen-, sulfur-, and nitrogen and sulfur- deprivations on the C. reinhardtii starchless mutant cc5373-sta6. Specifically, it compares growth, lipid body accumulation, and expression levels of acetyl-CoA carboxylase (ACC) and phosphoenolpyruvate carboxylase (PEPC). Results Among nutrient-deprived cells, TAP-S cells showed significantly higher total chlorophyll, cell density, and protein content at day 6 (p < 0.05). Confocal analysis showed a significantly higher number of lipid bodies in cells subjected to nutrient deprivation than in the control over the course of six days; N deprivation for six days significantly increased the size of lipid bodies (p < 0.01). In comparison with the control, significantly higher ACC expression was observed after 8 and 24 h of NS deprivation and only after 24 h with N deprivation. On the other hand, ACC and PEPC expression at 8 and 24 h of S deprivation was not significantly different from that in the control. A significantly lower PEPC expression was observed after 8 h of N and NS deprivation (p < 0.01), but a significantly higher PEPC expression was observed after 24 h (p < 0.01). Conclusions Based on our findings, it would be optimum to cultivate cc5373-sta6 cells in nutrient deprived conditions (-N, -S or –NS) for four days; whereby there is cell growth, and both a high number of lipid bodies and a larger size of lipid bodies produced

Mini-review: toxicity of mercury as a consequence of enzyme alteration

Mercury, in both its elemental and bonded states, is noted for its negative effects on biological organisms. Recent anthropogenic and environmental disasters have spurred numerous comparative studies. These studies attempted to detail the biochemical implications of mercury ingestion, in low, persistent concentrations as well as elevated acute dosages. The studies propose models for mercuric action on healthy cells; which is centered on the element’s disruption of key enzymatic processes at deposition sites. Mercury’s high affinity for the sulfhydryl moieties of enzyme catalytic sites is a common motif for enzyme inactivation. These permanent covalent modifications inactivate the enzyme, thereby inducing devastating effects on an organism’s metabolic functions. Mercury has been shown to be highly nonspecific in its binding to sulfhydryl moieties, and highly varied in terms of how it is encountered by living organisms. This review focuses on mercury’s effects on a wide swath of enzymes, with emphasis on how these alterations deleteriously affect several metabolic pathways

Identification and characterisation of a novel inorganic carbon acquisition gene, CIA7, from an insertional mutant of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a unicellular eukaryotic alga which possesses a CO2-concentrating mechanism (CCM) that enables it to grow at low CO2 concentrations. Previously, insertional mutants were generated to enable isolation of inorganic carbon transporters and other proteins that might be essential for a functional CCM. These mutants have an antibiotic resistance gene that encodes a protein that binds to Zeocin inhibiting Zeocin\u27s DNA strand cleavage activity. The DNA flanking the BleR insert of one of the high CO2 requiring strains, named cia7, was cloned with inverse-PCR and sequenced. Sequence analysis showed homology to conserved bacterial proteins of unknown function, but there were no ESTs in this region of the genome. However, the presence of a gene was established by PCR and RLM-RACE. CIA7 was found to have four exons and the BleR insert was in the fourth exon. CIA7 encodes a protein of 104 amino acids with a calculated molecular mass of 11.3:kDa. Based on the ChloroP prediction program, the protein is predicted to have a chloroplast targeting signal. Complementation analyses results showed possible partially rescued mutants, and RNAi showed several transformants with a sick on low CO2 phenotype with reduced expression of CIA7. These results suggest that CIA7 is a gene that facilitates growth in C. reinhardtii under low CO2 conditions. One possible role of CIA7 would be in the delivery or storage of a metal ion. It may play a potential role as either a domain of a metal transporter or as a metallochaperone. © CSIRO 2008

Learning tacit knowledge in life science graduate programs in Taiwan

In this paper we describe preliminary results of a three-year project that examines the enculturation of doctoral students in life science programs in Taiwan, Japan, and Singapore. The purpose of the study is to examine how doctoral students in life science enrolled at universities in these three countries learn to become scientists and how information and communication technologies affect such processes. The project is in its first year, and we completed data collection in Taiwan during the summer of 2009. Data was collected using quantitative surveys, qualitative interviews, and time-diaries from advisors and doctoral students in life science programs in three Taiwanese universities. Preliminary results show that current students tend to have problems related to too great a reliance on computers, kits, and the Internet, and as a result, they fail to assimilate tacit knowledge that is invaluable in becoming the next generation of scientists

Secondary metabolites, antibacterial and antioxidant properties of the leaf extracts of Acacia rigidula benth. and Acacia berlandieri benth.

The use of plants as sources for novel antimicrobial as well as antioxidant agents offers advantages. Plants are readily accessible and inexpensive, extracts or compounds from plant sources often demonstrate high level of biological activities. Previous studies have reported antibacterial and antifungal activities within the Fabaceae family that included Acacia species. This study aims to determine presence of antibacterial activity, antioxidant activity, and the secondary metabolites of sequential solvent extracts (acetone, methanol, and acetic acid) of Acacia berlandieri and Acacia rigidula leaves. The antibacterial activity was investigated using a disc diffusion assay. The ferric thiocyanate method was used to assess the ability of all extracts to prevent oxidation. Qualitative phytochemical tests, NMR, IR, and UV–Vis spectroscopy were done to identify potential secondary metabolites. P. alcalifaciens (p \u3c 0.001), E. faecalis (p \u3c 0.01), S. aureus (p \u3c 0.001), and Y. enterocolitica (p \u3c 0.001) were significantly inhibited by A. rigidula extracts when compared to A. berlandieri extracts. A. rigidula’s acetone extract exhibited the significantly (p \u3c 0.001) highest inhibition of peroxidation, 42%. Qualitative phytochemical tests showed positive results for presence of phenols, flavonoids, saponins, terpenes and tannins. NMR, IR, and UV–Vis spectroscopy revealed chemical structures found in flavonoids, saponins, terpenes and tannins, supporting the results of qualitative phytochemical tests. A. berlandieri and A. rigidula leaf extracts have revealed presence of medicinally valued bioactive components. The results of this study provide a basis for further investigations of the A. rigidula leaf extracts. A. rigidula leaf extracts have the potential to serve as a source of novel antimicrobial and antioxidant agents. Graphic abstract: [Figure not available: see fulltext.