Aquatic photosynthetic microorganisms account for almost 50% of the world's photosynthesis (19). These organisms face several challenges in acquiring CO 2 from the environment. The first challenge is presented by the properties of ribulose bisphosphate carboxylase-oxygenase (Rubisco). Rubisco is an unusually slow enzyme with a low affinity for CO 2 . At atmospheric levels of CO 2 , Rubisco can function at only about 25% of its catalytic capacity because the concentration of dissolved CO 2 is less than the K m (CO 2 ) of Rubisco and due to the relatively high concentration of O 2 which competes with CO 2 . A second challenge these organisms face is that the diffusion of CO 2 in an aqueous solution is 10,000 times slower than the diffusion of CO 2 in air. Thus, the ability to scavenge CO 2 as quickly as it becomes available is highly advantageous to aquatic photosynthetic organisms. Third, algae often experience significant fluctuations in inorganic carbon (C i ϭ CO 2 ϩ HCO 3 Ϫ ) levels and pH, which change the availability of CO 2 and HCO 3 Ϫ for photosynthesis. At an acidic pH, the vast majority of C i is in the form of CO 2 , while at an alkaline pH,

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.

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO2 and HCO3–. Aquatic photosynthetic organisms have evolved different forms of CO2-concentrating mechanisms to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CO2-concentrating mechanisms is the critical roles played by various specially localized extracellular and intracellular CAs. There are three evolutionarily unrelated CA families designated α-, β-, and γ-CA. In the green alga, Chlamydomonas reinhardtii Dangeard, eight CAs have now been identified, including three α-CAs and five β-CAs. In addition, C. reinhardtii has another CA-like gene, Glp1 that is similar to known γ-CAs. To characterize these different CA isoforms, some of the CA genes have been overexpressed to determine whether the proteins have CA activity and to generate antibodies for in vivo immunolocalization. The CA proteins Cah3, Cah6, and Cah8, and the γ-CA-like protein, Glp1, have been overexpressed. Cah3, Cah6, and Cah8 have CA activity, but Glp1 does not. At least two of these proteins, Cah3 and Cah6, are localized to the chloroplast. Using immunolocalization and sequence analyses, we have determined that Cah6 is located to the chloroplast stroma and confirmed that Cah3 is localized to the chloroplast thylakoid lumen. Activity assays show that Cah3 is 100 times more sensitive to sulfonamides than Cah6. We present a model on how these two chloroplast CAs might participate in the CO2-concentrating mechanism of C. reinhardtii. Key words: carbonic anhydrase, CO2-concentrating mechanism, Chlamydomonas, immunolocalization.

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.

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.

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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 negativebinomial 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. V C 2014 by The International Union of Biochemistry and Molecular Biology, 42(4):305-322, 2014.

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 < 0.001), E. faecalis (p < 0.01), S. aureus (p < 0.001), and Y. enterocolitica (p < 0.001) were significantly inhibited by A. rigidula extracts when compared to A. berlandieri extracts. A. rigidula’s acetone extract exhibited the significantly (p < 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