Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications.

An outbreak of pellagra related to changes in dietary niacin among Mozambican refugees in Malawi.

Between February and October 1990, 18,276 cases of pellagra dermatitis (due to niacin deficiency) were reported among 285,942 Mozambican refugees in Malawi. Overall, 6.3% of the refugee population developed pellagra and the attack rate was 7.8 times higher among women than men. This outbreak followed a 5-month cessation of groundnut distribution (the major source of niacin) to refugees. A matched-pair case-control study confirmed the protective role of the daily consumption of groundnuts (Odds Ratio [OR] = 0.08), as well as the independent role of garden ownership (OR = 0.34), and home maize milling (OR = 0.3). Recommended corrective action included early case finding and treatment, distribution of niacin tablets, prompt identification of groundnut supply on the world market, fortification with niacin of the food ration and diversification of the food basket through access to local markets.

PIP: Between February and October 1990, health workers in Malawi noted 18,276 cases of pellagra among 285,942 Mozambican refugees. This represented a significant increase in pellagra cases (compared with just 1169 cases in 1989). 5 months before each outbreak, the UN High Commission for Refugees and the World Food Program could not obtain groundnuts, a source of niacin, to include in food rations. The food ration distributed to refugees had an average of just 4 mg available niacin equivalent (or 2 mg/1000 kcal) which was considerably less than the recommended daily allowance of 6.6 mg/1000 kcal. The overall attack rate stood at 6.4% (4.9-13.2%. It was higher among refugees living in camps than it was among those living in Malawian villages near the border (10.1% vs. 0.8%). The attack rate was 7.8 times higher in females than males (6.1/1000 vs. 0.78/1000). It was lowest among children under 5 years old (1.7% vs. 7.5% for = or 5 year olds). No infant had pellagra. Researchers compared 126 pellagra cases with 126 controls. The conditional logistic regression indicated that pellagra cases were less likely to eat groundnuts and fish at least once a day within the last 6 months (odds ratio [OR] = .07 and .56, respectively). They tended not to have a garden (OR = .32) and to mill maize at home (OR = .26). Thus, eating groundnuts, milling maize at home, and garden ownership protected the refugees from developing pellagra. In August 1990, relief workers distributed niacin tablets to refugees. The health workers recommended other corrective actions such as early case finding and treatment, identification of groundnut supply on the world market, and diversification of the food basket through access to local markets.