Ruby Ynalvez

Proposed Carbon Dioxide Concentrating Mechanism in Chlamydomonas reinhardtii

Aquatic photosynthetic microorganisms account for almost 50% of the worlds photosynthesis ([19][1]). These organisms face several challenges in acquiring CO2 from the environment. The first challenge is presented by the properties of ribulose bisphosphate carboxylase-oxygenase (Rubisco). Rubisco is

Identification of a New Chloroplast Carbonic Anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrases (CA) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO2. The three evolutionarily unrelated families of CAs are designated α-, β-, and γ-CA. Aquatic photosynthetic organisms have evolved different forms of CO2 concentrating mechanisms (CCMs) to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CCMs is the critical roles played by various specially localized extracellular and intracellular CAs. Five CAs have previously been identified in Chlamydomonas reinhardtii, a green alga with a well-studied CCM. Here we identify a sixth gene encoding a β-type CA. This new β-CA, designated Cah6, is distinct from the two mitochondrial β-CAs in C. reinhardtii. Nucleotide sequence data show that the Cah6 cDNA contains an open reading frame encoding a polypeptide of 264 amino acids with a leader sequence likely targeting the protein to the chloroplast stroma. We have fused the Cah6 open reading frame to the coding sequence of maltose-binding protein in a pMal expression vector. The purified recombinant fusion protein is active and was used to partially characterize the Cah6 protein. The purified recombinant fusion protein was cleaved with protease Factor Xa to separate Cah6 from the maltose-binding protein and the purified Cah6 protein was used to raise an antibody. Western blots, immunolocalization studies, and northern blots collectively indicated that Cah6 is constitutively expressed in the stroma of chloroplasts. A possible role for Cah6 in the CCM of C. reinhardtii is proposed.

Identification and characterization of two closely related β‐carbonic anhydrases from Chlamydomonas reinhardtii

Aquatic photosynthetic organisms such as the green alga Chlamydomonas reinhardtii respond to low-CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Important components of the CCM are the carbonic anhydrases (CAs), zinc metalloenzymes that catalyze the interconversion of CO(2) and HCO(-)(3). Six CAs have previously been identified in C. reinhardtii. Here, we identify and characterize two additional beta-type CAs. These two CAs are closely related beta-type CAs and have been designated as CAH7 and CAH8. Conceptual translation shows that CAH7 and CAH8 encode proteins of 399 and 333 amino acids, respectively, and they contain targeting sequences. An unusual characteristic of these two CAs is that they have carboxy-terminal extensions containing a hydrophobic sequence. Both these CAs are constitutively expressed at the transcript and protein level. The CAH7 and CAH8 open reading frames were cloned in the overexpression vector pMal-c2x and expressed as recombinant proteins. Activity assays showed that CAH7 and CAH8 are both active CAs. Antibodies were raised against both CAH7 and CAH8, and immunolocalization studies showed that CAH8 was localized in the periplasmic space. A possible role for CAH8 in the inorganic carbon acquisition by C. reinhardtii is discussed.

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.

A mutant of Chlamydomonas reinhardtii that cannot acclimate to low CO2 conditions has an insertion in the Hdh1 gene

Photosynthetic microorganisms must acclimate to environmental conditions, such as low CO2 environments or high light intensities, which may lead to photo-oxidative stress. In an effort to understand how photosynthetic microorganisms acclimate to these conditions, Chlamydomonas reinhardtii was transformed using the BleR cassette, selected for Zeocin resistance and screened for colonies that showed poor growth at low CO2 levels. One of the insertional mutants obtained, named slc-230, was shown to have a BleR insert in the first exon of Hdh1, a novel, single copy gene. The predicted Hdh1 gene product has similarity to bacterial haloacid dehalogenase-like proteins, a protein family that includes phosphatases and epoxide hydrolases. In addition, Hdh1 is predicted to be localised to the chloroplast or mitochondria in C. reinhardtii. It was found that a genomic copy of wild type Hdh1 can complement slc-230.

Research experiences and mentoring practices in selected east asian graduate programs: Predictors of research productivity among doctoral students in molecular biology

Although doctoral mentors recognize the benefits of providing quality advisement and close guidance, those of sharing project management responsibilities with mentees are still not well recognized. We observed that mentees, who have the opportunity to co‐manage projects, generate more written output. Here we examine the link between research productivity, doctoral mentoring practices (DMP), and doctoral research experiences (DRE) of mentees in programs in the non‐West. Inspired by previous findings that early career productivity is a strong predictor of later productivity, we examine the research productivity of 210 molecular biology doctoral students in selected programs in Japan, Singapore, and Taiwan. Using principal component (PC) analysis, we derive two sets of PCs: one set from 15 DMP and another set from 16 DRE items. We model research productivity using Poisson and negative‐binomial regression models with these sets as predictors. Our findings suggest a need to re‐think extant practices and to allocate resources toward professional career development in training future scientists. We contend that doctoral science training must not only be an occasion for future scientists to learn scientific and technical skills, but it must also be the opportunity to experience, to acquire, and to hone research management skills.

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

SUMMARY OBJECTIVE This study examines the association of childrens (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 childrens social environment and ICT ownership predict BMIp; specifically, number of siblings (M2: β = -0.34, p-value < .001), and ownership of iPod/MP3 players (M2: β = 0.33, p-value < .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.

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 Zeocins 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.

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.