ANTARCTIC DISTRIBUTION,PIGMENT AND LIPID COMPOSITION,AND MOLECULAR IDENTIFICATION OF THE BRINE DINOFLAGELLATE POLARELLA GLACIALIS (DINOPHYCEAE)1

Polarella glacialis (Montresor et al.) was identified in Davis Station sea ice by morphological and DNA sequence comparison of cultures with those of the authentic strain P. glacialis CCMP 1383 isolated from McMurdo Sound. Cells and cysts of the Davis isolate (FL1B) were morphologically indistinguishable from P. glacialis, and comparison of the large subunit rDNA of both cultures demonstrated only 0.2% sequence divergence over 1366 base pairs. The photosynthetic pigments of P. glacialis (strains FL1B and CCMP 1383) were typical of dinoflagellates, with peridinin (contributing up to 31%) as the major accessory pigment. Extremely high levels of polyunsaturated fatty acids (PUFA, up to 76.3%) were characteristic of P. glacialis isolate FL1B. The high PUFA concentration of this species is thought to be an adaptation to survive the cold temperatures of the upper fast ice. The sterol profile of FL1B was atypical of dinoflagellates, with 4‐desmethylsterols (up to 79%) in greater abundance than 4α‐methyl sterols (up to 24%). 27‐Nor‐24‐methylcholest‐5,22E‐dien‐3β‐ol was identified as the principle sterol in P. glacialis, contributing up to 64% of the total sterol composition.     

Iron Stress and Pyoverdin Production by a Fluorescent Pseudomonad in the Rhizosphere of White Lupine (Lupinus albus L.) and Barley (Hordeum vulgare L.)

Induction of high-affinity iron transport during root colonization by Pseudomonas fluorescens Pf-5 (pvd-inaZ) was examined in lupine and barley growing in microcosms. P. fluorescens Pf-5 (pvd-inaZ) contains a plasmid carrying pvd-inaZ; thus, in this strain, ice nucleation activity is regulated by pyoverdin production. Lupine or barley plants were grown for 18 or 8 days, respectively, in soil amended with 2% calcium carbonate and inoculated with P. fluorescens Pf-5 (pvd-inaZ) at a density of 4 x 10(sup8) CFU g (dry weight) of soil(sup-1). A filter paper blotting technique was used to sample cells from the rhizosphere in different root zones, and then the cells were resuspended for enumeration and measurement of ice nucleation activity. The population density of P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere decreased by one order of magnitude in both lupine and barley over time. The ice nucleation activity ranged from -3.4 to -3.0 log ice nuclei CFU(sup-1) for lupine and -3.0 to -2.8 log ice nuclei CFU(sup-1) for barley, was similar in all root zones, and did not change over time. An in vitro experiment was conducted to determine the relationship between ice nucleation activity and pyoverdin production in P. fluorescens Pf-5 (pvd-inaZ). An ice nucleation activity of approximately -3.0 log ice nuclei CFU(sup-1) was measured in the in vitro experiment at 25 to 50 (mu)M FeCl(inf3). By using the regression between ice nucleation activity and pyoverdin production determined in vitro and assuming a P. fluorescens Pf-5 (pvd-inaZ) population density of 10(sup8) CFU g of root(sup-1), the maximum possible pyoverdin accumulation by P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere was estimated to be 0.5 and 0.8 nmol g of root(sup-1) for lupine and barley, respectively. The low ice nucleation activity measured in the rhizosphere suggests that nutritional competition for iron in the rhizosphere may not be a major factor influencing root colonization by P. fluorescens Pf-5 (pvd-inaZ).     

Tracing carbon flow in an arctic marine food web using fatty acid-stable isotope analysis

Global warming and the loss of sea ice threaten to alter patterns of productivity in arctic marine ecosystems because of a likely decline in primary productivity by sea ice algae. Estimates of the contribution of ice algae to total primary production range widely, from just 3 to >50%, and the importance of ice algae to higher trophic levels remains unknown. To help answer this question, we investigated a novel approach to food web studies by combining the two established methods of stable isotope analysis and fatty acid (FA) analysis--we determined the C isotopic composition of individual diatom FA and traced these biomarkers in consumers. Samples were collected near Barrow, Alaska and included ice algae, pelagic phytoplankton, zooplankton, fish, seabirds, pinnipeds and cetaceans. Ice algae and pelagic phytoplankton had distinctive overall FA signatures and clear differences in delta(13)C for two specific diatom FA biomarkers: 16:4n-1 (-24.0+/-2.4 and -30.7+/-0.8 per thousand, respectively) and 20:5n-3 (-18.3+/-2.0 and -26.9+/-0.7 per thousand, respectively). Nearly all delta(13)C values of these two FA in consumers fell between the two stable isotopic end members. A mass balance equation indicated that FA material derived from ice algae, compared to pelagic diatoms, averaged 71% (44-107%) in consumers based on delta(13)C values of 16:4n-1, but only 24% (0-61%) based on 20:5n-3. Our estimates derived from 16:4n-1, which is produced only by diatoms, probably best represented the contribution of ice algae relative to pelagic diatoms. However, many types of algae produce 20:5n-3, so the lower value derived from it likely represented a more realistic estimate of the proportion of ice algae material relative to all other types of phytoplankton. These preliminary results demonstrate the potential value of compound-specific isotope analysis of marine lipids to trace C flow through marine food webs and provide a foundation for future work.     

Vitrification enhancement by synthetic ice blocking agents

Small concentrations of the synthetic polymer polyvinyl alcohol (PVA) were found to inhibit formation of ice in water/cryoprotectant solutions. Ice inhibition improved with decreasing molecular weight. A PVA copolymer of molecular weight 2 kDa consisting of 20% vinyl acetate was found to be particularly effective. PVA copolymer concentrations of 0.001, 0.01, 0.1, and 1% w/w decreased the concentration of glycerol required to vitrify in a 10-ml volume by 1, 3, 4, and 5% w/w, respectively. Dimethyl sulfoxide concentrations required for vitrification were also reduced by 1, 2, 2, and 3% w/w, respectively. Crystallization of ice on borosilicate glass in contact with cryoprotectant solutions was inhibited by only 1 ppm of PVA copolymer. Devitrification of ethylene glycol solutions was also strongly inhibited by PVA copolymer. Visual observation and differential scanning calorimeter data suggest that PVA blocks ice primarily by inhibition of heterogeneous nucleation. PVA thus appears to preferentially bind and inactivate heterogeneous nucleators and/or nascent ice crystals in a manner similar to that of natural antifreeze proteins found in cold-hardy fish and insects. Synthetic PVA-derived ice blocking agents can be produced much less expensively than antifreeze proteins, offering new opportunities for improving cryopreservation by vitrification.     

Determination of the degree of methyl esterification of pectins in small samples by selective reduction of esterified galacturonic acid to galactose

A procedure for the determination of the degree of methyl esterification of pectin in virtually any sample is described. Samples were dissolved or suspended in 1 M imidazole buffer, pH 7.0, cooled on ice, and reduced with sodium borohydride. Quantitative reduction of samples was accomplished after 1 h using at least 20 mg sodium borohydride/mg sample. The degree of methyl esterification was determined by either the increase in galactose content as determined by GLC of the sugar or by the change in galacturonic acid content by colorimetric uronic acid analyses. Sample requirements were at least as low as 100 micrograms per determination by GLC or 2 to 3 mg per determination by colorimetric uronic acid analysis compared to 5 mg or more per determination for other published procedures. The degrees of methyl esterification determined by the methods described have compared very favorably with those determined by established methods.     

Winter behaviour of juvenile Atlantic salmon Salmo salar L. in experimental stream channels: effect of substratum size and full ice cover on spatial distribution and activity pattern

Activity and choice of areas offering different cover (substratum or surface ice) for juvenile Atlantic salmon Salmo salar were studied in experimental stream channels during winter. Channels were completely ice covered between December and March. During this period, the ice thickness increased from 50 to 300 mm after which 50% of the ice was experimentally removed and followed by c. 2·5-fold increase in discharge to simulate the effects of spring flood. Large substrata provided preferred habitats but areas with small substratum sizes were also used when full surface ice provided above-stream cover and the stream discharge was relatively low. The fish remained nocturnal throughout the study but the level of day activity significantly increased as the surface ice became thicker. Maximum movement distance during a 24 h period and homing-at-dawn behaviour remained at a constant level throughout the main winter, but significantly changed during the simulated spring flood (mean ± s . e . maximum extent of movements within 24 h increased from 1·1 ± 0·1 to 3·0 ± 0·5 m; homing behaviour decreased from the highest level of 89·3 to 34·6% during spring flood). Overwinter survival was high (92·9%). Relative mass increase during the study ranged from –8·3 to 28·5%, and 84% of the juvenile Atlantic salmon gained mass. The highest rates of mass increase were associated with frequent movements between areas of different substratum size. The results indicate that during winter: (1) Atlantic salmon parr preferred large substratum cover compared with surface ice cover at the fish densities studied here, (2) juvenile Atlantic salmon were predominantly nocturnal but diurnal activity increased as surface ice became thicker and (3) increase in water discharge during spring altered the behaviour of juvenile Atlantic salmon and may have caused additional habitat shifts.     

The role of ice nucleation active bacteria in supercooling of citrus tissues

The frost sensitivity of Citrus sinensis in relation to the presence of biogenic ice nuclei was studied. In commercially managed citrus groves the ice nucleation active (INA) bacterium Pseudomonas syringae reached 6 × 10⁴ colony forming units (CFU) leaf⁻¹, a population sufficiently high to catalyze ice formation. However, a transient loss of bacterial nucleation activity was noticeable at subzero field temperatures, followed by resumption as temperatures rose. This loss was apparently due to a temporary transition of INA to ice nucleation inactive (INI) bacteria. Field application of Bordeaux mixture, copper hydroxide, streptomycin, and 2-hydroxypropylmethanethiolsulfonate (HPMTS), resulted in reduction of INA bacterial populations to detectability (≤ 10² CFU leaf⁻¹) limits. However, the corresponding reduction in ice nucleation events in treated samples as compared to controls at nucleation temperature ≥−3°C was not as dramatic. It ranged from approximately 7% in samples treated with the bactericide HPMTS, to 35% in samples treated with chemicals possessing combined bactericidal - fungicidal action (coppers). Since a quantitative relationship exists between ice nucleation events on individual leaves and the INA bacterial populations harbored by these leaves, these results suggest the co-existence of a bacterial and a proteinaceous, yet non-bacterial ice nucleating source in citrus, both active at ≥−3°C.     

Cervical dilatation with prostaglandin analogues prior to vaginal termination of first trimester pregnancy in nulliparous patients

Dilatation of the cervix with prostaglandin analogues prior to vaginal termination of pregnancy was attempted in 125 nulliparous women in the first trimester of pregnancy. The patients were divided into five groups (25 in each group) and given a single extra-amniotic dose of one of the following prostaglandin analogues 14–16 hours prior to the evacuation of the uterus by vacuum aspiration. (Group A) 15 (S) 15 methyl PGE₂ (free acid); (Group B) 15 (S) 15 methyl PGE₂ methyl ester; (Group C) 15 (S) 15 methyl PGF_2α (free acid); (Group D) 15 (S) 15 methyl PGF_2α methyl ester and(Group E) a mixture of 15 (S) 15 methyl PGE₂ methyl ester and 15 (S) 15 methyl PGF_2α methyl ester. Evacuation of the uterus without mechanical dilatation of the cervix was possible in 111 (90%) of the patients. In an additional 10 patients (8%) there was some degree of cervical dilatation and further mechanical dilatation could be performed easily. With the combination of 15 (S) 15 methyl PGE₂ methyl ester and 15 (S) 15 methyl PGF_2α methyl ester the incidence of gastrointestinal side effects and pyrexia were considerably reduced.     

The Southern Ocean ecosystem under multiple climate change stresses ‐ an integrated circumpolar assessment

A quantitative assessment of observed and projected environmental changes in the Southern Ocean (SO) with a potential impact on the marine ecosystem shows: (i) large proportions of the SO are and will be affected by one or more climate change processes; areas projected to be affected in the future are larger than areas that are already under environmental stress, (ii) areas affected by changes in sea‐ice in the past and likely in the future are much larger than areas affected by ocean warming. The smallest areas (<1% area of the SO) are affected by glacier retreat and warming in the deeper euphotic layer. In the future, decrease in the sea‐ice is expected to be widespread. Changes in iceberg impact resulting from further collapse of ice‐shelves can potentially affect large parts of shelf and ephemerally in the off‐shore regions. However, aragonite undersaturation (acidification) might become one of the biggest problems for the Antarctic marine ecosystem by affecting almost the entire SO. Direct and indirect impacts of various environmental changes to the three major habitats, sea‐ice, pelagic and benthos and their biota are complex. The areas affected by environmental stressors range from 33% of the SO for a single stressor, 11% for two and 2% for three, to <1% for four and five overlapping factors. In the future, areas expected to be affected by 2 and 3 overlapping factors are equally large, including potential iceberg changes, and together cover almost 86% of the SO ecosystem.     

Functional biodiversity of lipids in Antarctic zooplankton: Calanoides acutus, Calanus propinquus, Thysanoessa macrura and Euphausia crystallorophias

Zooplankton samples were collected in January 1993 off Dronning Maud Land along a transect from open waters to the marginal ice zone close to the Antarctic ice shelf. Thysanoessa macrura was caught in open waters while Calanoides acutus and Calanus propinquus were mainly sampled between ice floes in the marginal ice zone. The “ice-krill”Euphausia crystallorophias was found over the shelf directly associated with ice floes. T. macrura had a lipid content up to 36% of its dry weight with the dominant lipid class, wax ester, accounting for 45–50% of the total lipid. The predominance of 18:1 fatty alcohols is the striking characteristic of the wax esters. Small specimens of E. crystallorophias had lipid levels up to 26% of their dry weight with, unexpectedly, triacylglycerols being the dominant lipid (up to 41% of total lipid). The small levels of wax esters in these animals (3–6% of total lipid) had phytol as a major constituent. Large specimens of E. crystallorophias had up to 34% of their dry weight as lipid, with wax esters (47% of total lipid) dominated by 16:0 and 14:0 fatty alcohols as the major lipid. Calanus propinquus had lipid levels of up to 34% of their dry weight, with triacylglycerols (up to 63% of total lipid) being the dominant lipid. High levels of 22:1 (n-9) fatty acid were present in the triacylglycerols. Calanoides acutus had lipid levels up to 35% of the dry weight with wax esters accounting for up to 83% of total lipid. High levels of (n-3) polyunsaturated fatty acids were recorded with 20:5(n-3), 22:6(n-3) and 18:4(n-3) being the dominant moieties. On the basis of their lipid compositions we deduce that: (1) Calanoides acutus is the strictest herbivore among the four species studied, heavily utilizing the typical spring bloom; (2) T. macrura is essentially omnivorous, probably utilizing the less defined bloom situations found in oceanic waters; (3) E. crystallorophias is an omnivore well adapted to utilize both a bloom situation and to feed on ice algae and micro-zooplankton associated with the ice; (4) Calanus propinquus seems to be the most opportunistic feeder of the four species studied, probably grazing heavily on phytoplankton during a bloom and, during the rest of the year, feeding on whatever material is available, including particulates, flagellates and other ice-associated algae. We conclude that the different biochemical pathways generating large oil reserves of different compositions, enabling species to utilize different ecological niches, are major determinants of biodiversity in polar zooplankton. Accepted: 22 June 1998     

Environmental and developmental regulation of the wound-induced cell wall protein WI12 in the halophyte ice plant

A wounded gene WI12 was used as a marker to examine the interaction between biotic stress (wounding) and abiotic stress (high salt) in the facultative halophyte ice plant (Mesembryanthemum crystallinum). The deduced WI12 amino acid sequence has 68% similarity to WUN1, a known potato (Solanum tuberosum) wound-induced protein. Wounding, methyl jasmonate, and pathogen infection induced local WI12 expression. Upon wounding, the expression of WI12 reached a maximum level after 3 h in 4-week-old juvenile leaves, whereas the maximum expression was after 24 h in 8-week-old adult leaves. The temporal expression of WI12 in salt-stressed juvenile leaves was similar to that of adult leaves. The result suggests that a salt-induced switch from C3 to Crassulacean acid metabolism has a great influence on the ice plant's response to wounding. The expression of WI12 and the accumulation of WI12 protein were constitutively found in phloem and in wounded mesophyll cells. At the reproductive stage, WI12 was constitutively found in petals and styles, and developmentally regulated in the placenta and developing seeds. The histochemical analysis showed that the appearance of WI12 is controlled by both environmental and developmental factors. Immunogold labeling showed WI12 preferentially accumulates in the cell wall, suggesting its role in the reinforcement of cell wall composition after wounding and during plant development.     

Increased Arctic sea ice drift alters adult female polar bear movements and energetics

Recent reductions in thickness and extent have increased drift rates of Arctic sea ice. Increased ice drift could significantly affect the movements and the energy balance of polar bears (Ursus maritimus) which forage, nearly exclusively, on this substrate. We used radio‐tracking and ice drift data to quantify the influence of increased drift on bear movements, and we modeled the consequences for energy demands of adult females in the Beaufort and Chukchi seas during two periods with different sea ice characteristics. Westward and northward drift of the sea ice used by polar bears in both regions increased between 1987–1998 and 1999–2013. To remain within their home ranges, polar bears responded to the higher westward ice drift with greater eastward movements, while their movements north in the spring and south in fall were frequently aided by ice motion. To compensate for more rapid westward ice drift in recent years, polar bears covered greater daily distances either by increasing their time spent active (7.6%–9.6%) or by increasing their travel speed (8.5%–8.9%). This increased their calculated annual energy expenditure by 1.8%–3.6% (depending on region and reproductive status), a cost that could be met by capturing an additional 1–3 seals/year. Polar bears selected similar habitats in both periods, indicating that faster drift did not alter habitat preferences. Compounding reduced foraging opportunities that result from habitat loss; changes in ice drift, and associated activity increases, likely exacerbate the physiological stress experienced by polar bears in a warming Arctic.     

Biosynthesis of phytoquinones. Incorporation of l-[Me-14C3H]methionine into terpenoid quinones and chromanols in maize shoots

1. Radioactivity from l-[Me-(14)C,(3)H]methionine is incorporated into phylloquinone, plastoquinone, gamma-tocopherol, alpha-tocopherol, alpha-tocopherolquinone and ubiquinone in maize shoots. 2. Comparative studies with other terpenoids (squalene and beta-carotene) and chemical degradation of selected quinones (ubiquinone and plastoquinone) established that all the radioactivity is confined to nuclear methyl substituents. 3. In ubiquinone 76% of the radioactivity is in the methoxyl groups and 24% in the ring C-methyl group. 4. Taking the phytosterols as an internal reference and accepting the atomic ratio of (14)C/(3)H transferred from l-[Me-(14)C,(3)H]methionine to the supernumerary group at C(24) to be 1:2 the ratio of all the quinones and chromanols examined approached 1:3. After allowing for the fact that for plastoquinone, gamma-tocopherol, alpha-tocopherol and alpha-tocopherolquinone one nuclear methyl group is formed from the beta-carbon of tyrosine, these results show that one nuclear C-methyl group for phylloquinone, plastoquinone and gamma-tocopherol, two nuclear methyl groups for alpha-tocopherol and alpha-tocopherolquinone and one nuclear methyl and two methoxyl groups for ubiquinone are formed by the transfer of intact methyl groups from methionine. 5. From a comparison of the incorporation of (14)C radioactivity into these compounds it would appear that the methylation reactions involved in phylloquinone and plastoquinone biosynthesis take place in the chloroplast, whereas those involved with ubiquinone biosynthesis occur else-where within the cell.     

Isolation of chlorogenic acids and their derivatives from Stemona japonica by preparative HPLC and evaluation of their anti-AIV (H5N1) activity in vitro

Two chlorogenic acids and five chlorogenic acid derivatives were simultaneously separated and purified from Stemona japonica by preparative high-performance liquid chromatography. Five of the collected compounds were over 95% pure while the other two compounds were over 90% pure. Their structures were elucidated as 3-O-feruloylquinic acid (1), 4-O-feruloylquinic acid (2), methyl 3-O-feruloylquinate (3), methyl 5-O-caffeyolquinate (4), methyl 4-O-feruloylquinate (5), ethyl 3-O-feruloylquinate (6) and the new compound ethyl 4-O-feruloylquinate (7) by UV, NMR and ESI-MS. All compounds were obtained from Stemona species for the first time, however compounds 6 and 7 are believed to be artefacts from the ethanol extraction. The anti-AIV (H5N1) activities were evaluated by Neutral Red uptake assay. Compounds 3 and 4 exerted moderate inhibitory effect against AIV (H5N1) in vitro.     

Biomass,composition and activity of organism assemblages along a salinity gradient in sea ice subjected to river discharge in the Baltic Sea

A study was undertaken to examine the activity and composition of the seasonal Baltic Sea land-fast sea-ice biota along a salinity gradient in March 2003 in a coastal location in the SW coast of Finland. Using a multi-variable data set, the less well-known algal and protozoan communities, and algal and bacterial production in relation to the physical and chemical environment were investigated. Also, the first coincident measurements of bacterial production and dissolved organic matter (DOM) in a sea-ice system are reported. Communities in sea ice were clearly autotrophy-dominated with algal biomass representing 79% of the total biomass. Protozoa and rotifers made up 18% of biomass in the ice and bacteria only 3%. Highest biomasses were found in mid-transect bottom ice. Water column assemblages were clearly more heterotrophic: 39% algae, 12% bacteria and 49% for rotifers and protozoa. Few significant correlations existed between DOM and bacterial variables, reflecting the complex origin of ice DOM. Dynamics of dissolved organic carbon, nitrogen and phosphorus (DOC, DON and DOP) were also uncoupled. A functional microbial loop is likely to be present in the studied ice. Existence of an under-ice freshwater plume affects the ecosystem functioning: Under-ice water communities are influenced directly by river-water mixing, whereas the ice system seems to be more independent—the interaction mainly taking place through the formation of active bottom communities.     

Nucleation and growth of ice in deeply undercooled erythrocytes

Previous studies of the mechanism of freezing of erythrocytes in the absence of intracellular ice have been extended to define the catalytic sites responsible for promoting nucleation. The following aspects have been investigated: (1) the freeze propagation between undercooled erythrocytes, (2) the nucleation of ice in undercooled erythrocyte ghosts, and (3) the freezing behavior of undercooled hemoglobin solutions. The main findings are: (1) no cross-nucleation occurs between individual cells packed within the same emulsified water droplet; (2) the differential scanning calorimetric power-time curves of intact cells and ghosts are identical, indicating that hemoglobin does not affect ice nucleation; (3) the nucleation temperature of ice in an aqueous solution of hemoglobin (isolated from the cells) is substantially lower than that for the same solution when contained in the intact cell; (4) the threefold freeze concentration which accompanies the freezing of a 25% hemoglobin solution does not cause denaturation of the protein.     

Mutagenic and inactivating effects of methyl alkylaminosulfonates on Escherichia coli.

Methyl alkylamino methanesulfonates are mutagenic agents as shown by treating several strains of E. coli at pH 7. Methyl methylaminosulfonate (CH3-NH-SO3-CH3) was more efficient than methyl ethylaminosulfonate (C2H5-NH-SO3-CH3) which itself was more efficient than methyl isopropylaminosulfonate (C3H7-NH-SO3-CH3). Methyl methylaminosulfonate seemed to be at least as effective as methyl methanesulfonate (CH3-SO3-CH3). Methyl methylaminosulfonate produced a yield of up to 1% of auxotrophic mutants. All three new mutagens appeared to react according to the same mechanism by ester fission and methylation of nucleophilic groups as is known for methyl methanesulfonate. The reaction mechanism seems to be of the SN2 type.     

Insight into the binding of antifreeze proteins to ice surfaces via 13C spin lattice relaxation solid-state NMR

The primary sequences of type I antifreeze proteins (AFPs) are Ala rich and contain three 11-residue repeat units beginning with threonine residues. Their secondary structures consist of alpha-helices. Previous activity study of side-chain mutated AFPs suggests that the ice-binding side of type I AFPs comprises the Thr side chains and the conserved i + 4 and i + 8 Ala residues, where i indicates the positions of the Thrs. To find structural evidence for the AFP's ice-binding side, a variable-temperature dependent (13)C spin lattice relaxation solid-state NMR experiment was carried out for two Ala side chain (13)C labeled HPLC6 isoforms of the type I AFPs each frozen in H(2)O and D(2)O, respectively. The first one was labeled on the equivalent 17th and 21st Ala side chains (i + 4, 8), and the second one on the equivalent 8th, 19th, and 30th Ala side chains (i + 6). The two kinds of labels are on the opposite sides of the alpha-helical AFP. A model of Ala methyl group rotation/three-site rotational jump combined with water molecular reorientation was tested to probe the interactions of the methyl groups with the proximate water molecules. Analysis of the T(1) data shows that there could be 10 water molecules closely capping an i + 4 or an i + 8 methyl group within the range of van der Waals interaction, whereas the surrounding water molecules to the i + 6 methyl groups could be looser. This study suggests that the side of the alpha-helical AFP comprising the i + 4 and i + 8 Ala methyl groups could interact with the ice surface in the ice/water interface.