Production of the infant formula ingredient 1,3-olein-2-palmitin in Arabidopsis thaliana seeds

In human milk fat, palmitic acid (16:0) is esterified to the middle (sn-2 or β) position on the glycerol backbone and oleic acid (18:1) predominantly to the outer positions, giving the triacylglycerol (TG) a distinctive stereoisomeric structure that is believed to assist nutrient absorption in the infant gut. However, the fat used in most infant formulas is derived from plants, which preferentially esterify 16:0 to the outer positions. We have previously showed that the metabolism of the model oilseed Arabidopsis thaliana can be engineered to incorporate 16:0 into the middle position of TG. However, the fatty acyl composition of Arabidopsis seed TG does not mimic human milk, which is rich in both 16:0 and 18:1 and is defined by the high abundance of the TG molecular species 1,3-olein-2-palmitin (OPO). Here we have constructed an Arabidopsis fatty acid biosynthesis 1-1 fatty acid desaturase 2 fatty acid elongase 1 mutant with around 20% 16:0 and 70% 18:1 in its seeds and we have engineered it to esterify more than 80% of the 16:0 to the middle position of TG, using heterologous expression of the human lysophosphatidic acid acyltransferase isoform AGPAT1, combined with suppression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE. Our data show that oilseeds can be engineered to produce TG that is rich in OPO, which is a structured fat ingredient used in infant formulas.     

Tel2p, a regulator of yeast telomeric length in vivo, binds to single-stranded telomeric DNA in vitro

The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region of TG_1–3 repeats that acquire a single-stranded 3’ extension of the TG_1–3 strand at the end of S-phase. The length of these repeats is kept within a defined range by regulators such as the TEL2-encoded protein (Tel2p). Here we show that Tel2p can specifically bind to single-stranded TG_1–3. Tel2p binding produced several shifted bands; however, only the slowest migrating band contained Tel2p. Methylation protection and interference experiments as well as gel shift experiments using inosine-containing probes indicated that the faster migrating bands resulted from Tel2p-mediated formation of DNA secondary structures held together by G-G interactions. Tel2p bound to single-stranded substrates that were at least 19 bases in length and contained 14 bases of TG_1–3, and also to double-stranded/single-stranded hybrid substrates with a 3’ TG_1–3 overhang. Tel2p binding to a hybrid substrate with a 24 base single-stranded TG_1–3 extension also produced a band characteristic of G-G-mediated secondary structures. These data suggest that Tel2p could regulate telomeric length by binding to the 3’ single-stranded TG_1–3 extension present at yeast telomeres. Received: 12 November 1998; in revised form: 6 April 1999 / Accepted: 13 April 1999     

Study of the biological effects of theophylline on V79 cells: Viability,membrane permeability,and metabolic cooperation

The effect of theophylline, a specific inhibitor of phosphodiesterase, on gap junction-mediated intercellular communication between Chinese hamster V79 cells was examined. It was found that addition of theophylline to coculture of 6-thioguanine-resistant (TG^r) and 6-thioguanine-sensitive (TG^s) V79 cells significantly increased the recovery of TG^r cells. This finding indicates an inhibition of metabolic cooperation of V79 cells by theophylline. Theophylline was tested at concentrations <0.3 mg/ml, which were neither cytotoxic (after short or continuous exposure) nor inhibited the synthesis of DNA, RNA, and proteins. At the tested concentrations, no change was found in the membrane permeability of cells. Theophylline did not increase the incorporation of glucose into the cells.Abbreviations TG 6, thioguanine     

Differential postprandial incorporation of 20:5n-3 and 22:6n-3 into individual plasma triacylglycerol and phosphatidylcholine molecular species in humans

The mechanisms by which digested fat is absorbed and transported in the circulation are well documented. However, it is uncertain whether the molecular species composition of dietary fats influences the molecular species composition of meal-derived lipids in blood. This may be important because enzymes that remove meal-derived fatty acids from the circulation exhibit differential activities towards individual lipid molecular species. To determine the effect of consuming oils with different molecular compositions on the incorporation of 20:5n-3 and 22:6n-3 into plasma lipid molecular species. Men and women (18–30 years) consumed standardised meals containing 20:5n-5 and 22:6n-3 (total 450 mg) provided by an oil from transgenic Camelina sativa (CSO) or a blended fish oil (BFO) which differed in the composition of 20:5n-3 and 22:6n-3 – containing molecular species. Blood was collected during the subsequent 8 h. Samples were analysed by liquid chromatography-mass spectrometry. The molecular species composition of the test oils was distinct from the composition of plasma triacylglycerol (TG) or phosphatidylcholine (PC) molecular species at baseline and at 1.5 or 6 h after the meal. The rank order by concentration of both plasma PC and TG molecular species at baseline was maintained during the postprandial period. 20:5n-3 and 22:6n-3 were incorporated preferentially into plasma PC compared to plasma TG. Together these findings suggest that the composition of dietary lipids undergoes extensive rearrangement after absorption, such that plasma TG and PC maintain their molecular species composition, which may facilitate lipase activities in blood and/or influence lipoprotein structural stability and function.     

Stomatal response to blue light: water use efficiency in sugarcane and soybean*

Abstract. The significance of blue light-stimulated stomatal conductance for carbon assimilation (A), stomatal conductance (g), intercellular CO₂ (C_i), stomatal limitation of A (L), transpiration (E) and water use efficiency (W = A/E), was determined in a C₄ and a C₃ species. W and L were evaluated for steady-state gas exchange with constant, saturating red light (A_s, g_s, E_s), and for the integrated gas exchange above the steady state baseline induced by a single, brief pulse of blue light (A_p, g_p, E_p). Sugarcane (Saccharum spp. hybrid), a C₄ grass, and soybean (Glycine max) a C₃ dicot, were compared. Sugarcane exhibited typical C₄ behaviour, with A saturing at C_i of ca. 200 μmol mol^?1, compared to >500 μmol mol^?1 in soybean. Steady-state W was also considerably higher in sugarcane. The extent of stomatal opening in response to a blue light pulse, from baseline (g_s) to the maximum value of conductance during the opening response (g_m), was similar in the two species. More rapid opening and closing of stomata in sugarcane resulted in a smaller integrated magnitude of the conductance response (g_p) than in soybean. At the peak of the blue light response, both species exhibited similar levels of L. During the response to the pulse of blue light, A and C_i increased and L decreased to a greater extent in sugarcane than in soybean. As a result, the gas exchange attributed to the stomatal response to blue light exhibited a higher ratio of A_p to E_p (W_p) in sugarcane than in soybean. This W_p was lower in both species than was the W_s associated with the steady state gas exchange. The two species did not differ in the rate of induction of photosynthetic utilization of elevated C_i. The greater stimulation of A in sugarcane was attributed to its C₄ attributes of greater carboxylation efficiency (slope of the A versus C_i relationship), lower g_s and prevailing C_i,s, and greater L_s under steady-state red illumination. Despite saturation of A at low levels of C_i in C₄ species, the gas exchange attributed to the stomatal response to blue light decreased L and contributed considerably to carbon acquisition, while maintaining the high level of W associated with C₄ metabolism.     

‘Cross-talk’ between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the α-subunits of the stimulatory and inhibitory G-proteins of AC (G_sα and G_i−2α) in cultured prolactin-producing rat pituitary adenoma cells (GH₃ cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G_sα levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G_sα levels by more than 50% at all time-points. G_i−2α levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G_s protein α-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.     

薏苡仁油脂的微商热重分析

本文采用热重法比较了薏苡仁油与豆油的稳定性差异.结果表明,热稳定性及氧化稳定性顺序为氢化薏苡仁油>混合薏苡仁油>精炼薏苡仁油>精炼豆油,这主要取决于各自脂肪酸的组成.微商热重法简便、灵敏,还可用于鉴别薏苡仁油质量.     

Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

The Telomeric dG(GT)4G Sequence Can Adopt a Parallel-Stranded Double Helical Conformation

Abstract

Oligonucleotides 3′-d(GTGTGTGTGG)-L-d(GGTGTGTGTG)-3′ (hp-GT) and 3′- d(G^4STG^4STG^4STG^4STGG)-L-d(GGTGTGTGTG)-3′ (hp-SGT), (L=(CH₂CH₂O)₃), were shown by use of several optical techniques to form a novel parallel-stranded (ps) intramolecular double helix with purine-purine and pyrimidine-pyrimidine base pairing. The rotational relaxation time of hp-GT was similar to that of a 10-bp reference duplex, and the fraction of unpaired bases was determined to be ～ 7%, testifying to the formation of an intramolecular double helical hairpin by the sequence under the given experimental conditions. A quasi-two- state mode of ps-double helix formation was validated, yielding a helix-coil transition enthalpy of ?135±5 kJ/mol. The G-G and T·T (or ^4ST·T) base pair configurations and conformational parameters of the double helix were derived with molecular modeling by force field techniques. Repetitive d(GT) sequences are abundant in telomers of different genomes and in the regulatory regions of genes. Thus, the observed conformational potential of the repetitive d(GT) sequence may be of importance in the regulation of cell processes.     

Soybean leaf hydraulic conductance does not acclimate to growth at elevated [CO2] or temperature in growth chambers or in the field

Background and Aims

Leaf hydraulic properties are strongly linked with transpiration and photosynthesis in many species. However, it is not known if gas exchange and hydraulics will have co-ordinated responses to climate change. The objective of this study was to investigate the responses of leaf hydraulic conductance (K_leaf) in Glycine max (soybean) to growth at elevated [CO₂] and increased temperature compared with the responses of leaf gas exchange and leaf water status.

Methods

Two controlled-environment growth chamber experiments were conducted with soybean to measure K_leaf, stomatal conductance (g_s) and photosynthesis (A) during growth at elevated [CO₂] and temperature relative to ambient levels. These results were validated with field experiments on soybean grown under free-air elevated [CO₂] (FACE) and canopy warming.

Key results

In chamber studies, K_leaf did not acclimate to growth at elevated [CO₂], even though stomatal conductance decreased and photosynthesis increased. Growth at elevated temperature also did not affect K_leaf, although g_s and A showed significant but inconsistent decreases. The lack of response of K_leaf to growth at increased [CO₂] and temperature in chamber-grown plants was confirmed with field-grown soybean at a FACE facility.

Conclusions

Leaf hydraulic and leaf gas exchange responses to these two climate change factors were not strongly linked in soybean, although g_s responded to [CO₂] and increased temperature as previously reported. This differential behaviour could lead to an imbalance between hydraulic supply and transpiration demand under extreme environmental conditions likely to become more common as global climate continues to change.     

The Yeast Telomere Length Counting Machinery Is Sensitive to Sequences at the Telomere-Nontelomere Junction

Saccharomyces cerevisiae telomeres consist of a continuous 325 ± 75-bp tract of the heterogeneous repeat TG_1-3 which contains irregularly spaced, high-affinity sites for the protein Rap1p. Yeast cells monitor or count the number of telomeric Rap1p molecules in a negative feedback mechanism which modulates telomere length. To investigate the mechanism by which Rap1p molecules are counted, the continuous telomeric TG_1-3 sequences were divided into internal TG_1-3 sequences and a terminal tract separated by nontelomeric spacers of different lengths. While all of the internal sequences were counted as part of the terminal tract across a 38-bp spacer, a 138-bp disruption completely prevented the internal TG_1-3 sequences from being considered part of the telomere and defined the terminal tract as a discrete entity separate from the subtelomeric sequences. We also used regularly spaced arrays of six Rap1p sites internal to the terminal TG_1-3 repeats to show that each Rap1p molecule was counted as about 19 bp of TG_1-3 in vivo and that cells could count Rap1p molecules with different spacings between tandem sites. As previous in vitro experiments had shown that telomeric Rap1p sites occur about once every 18 bp, all Rap1p molecules at the junction of telomeric and nontelomeric chromatin (the telomere-nontelomere junction) must participate in telomere length measurement. The conserved arrangement of these six Rap1p molecules at the telomere-nontelomere junction in independent transformants also caused the elongated TG_1-3 tracts to be maintained at nearly identical lengths, showing that sequences at the telomere-nontelomere junction had an effect on length regulation. These results can be explained by a model in which telomeres beyond a threshold length form a folded structure that links the chromosome terminus to the telomere-nontelomere junction and prevents telomere elongation.     

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum

Summary To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a supersuppressor selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HAT^r) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT mutants — selected by their resistance to 6-thioguanine (TG^r) — and TK^- mutants — selected by their resistance to 5-bromodeoxyuridine (BrdUrd^r) — are HAT^s. Therefore, from HPRT^- TK^- double nonsense mutants, whose phenotype would be TG^r BrdUrd^r (HAT^s), simultaneous HPRT⁺ TK⁺ double phenotypic revertants could be obtained by selecting HAT^r (TG^s BrdUrd^s) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TG^r variants, 53 of which were HAT^s and the rest HAT^r. Among 36 TG^r (HAT^s) variants tested, 23 did not revert to HAT^r, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT^- nonsense mutants because they were very stringent (had less than 2% of wild-type [³H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrd^r) in 7 of these strains allowed us to isolate 29 TG^r BrdUrd^r (HAT^s) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HAT^r (TG^s BrdUrd^s) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.     

Yeast telomerase appears to frequently copy the entire template in vivo

Telomeres derived from the same formation event in wild type strains of Saccharomyces cerevisiae possess the same, precise TG_1–3 sequence for the most internal ~100 bp of the 250–350 bp TG_1–3 repeats. The conservation of this internal domain is thought to reflect the fact that telomere lengthening and shortening, and thus alteration of the precise TG_1–3 sequence, is confined to the terminal region of the telomere. The internal domains of telomeres from yku70Δ and tel1Δ mutants, whose entire telomeres are only ~100 bp, were examined by analyzing 5.1 kb of cloned TG_1–3 sequences from telomeres formed during transformation of wild type, yku70Δ and tel1Δ cells. The internal domains were 97–137 bp in wild type cells, 27–36 bp in yku70Δ cells and 7–9 bp in tel1Δ cells. These data suggest that the majority of the tel1Δ cell TG_1–3 repeats may be resynthesized during shortening and lengthening reactions while a portion of the yku70Δ cell telomeres are protected. TG_1–3 sequences are synthesized by telomerase repeatedly copying an internal RNA template, which introduces a sequence bias into TG_1–3 repeats. Analysis of in vivo-derived telomeres revealed that of the many possible high affinity binding sites for the telomere protein Rap1p in TG_1–3 repeats, only those consistent with telomere hybridization to the ACACAC in the 3′-region of the telomerase RNA template followed by copying of most of the template were present. Copies of the telomerase RNA template made up 40–60% of the TG_1–3 sequences from each strain and could be found in long, tandem repeats. The data suggest that in vivo yeast telomerase frequently allows telomeres to hybridize to the 3′-region of RNA template and copy most of it prior to dissociation, or that in vivo telomere processing events result in the production of TG_1–3 sequences that mimic this process.     

Differential Effects of Dietary Oils on Emotional and Cognitive Behaviors

Several dietary oils have been used preventatively and therapeutically in the setting of neurological disease. However, the mechanisms underlying their influence on brain function and metabolism remain unknown. It was investigated whether 3 types of dietary oils affected emotional behaviors in mice. Wild-type (WT) mice and sialyltransferase ST3Gal IV-knockout (KO) mice, which exhibit increased emotional and cognitive behaviors, were fed diets containing 20% dietary oils from post-weaning to adulthood. Mice were fed pellets made from control feed AIN93G powder containing 18% fish oil, soybean oil, or a mixture of 1-palmitoyl-2-oleoyl-3-palmitoyl glycerol (POP) and 1-stearoyl-2-oleoyl-3-stearoyl glycerol (SOS), plus 2% soybean oil. Once mice reached adulthood, they were subjected to fear conditioning test to measure cognitive anxiety and forced swim test to measure depression. WT mice fed the POP-SOS diet showed a 0.6-fold decrease in percent freezing with contextual fear compared with WT mice fed the control diet. KO mice fed the fish oil diet showed a 1.4-fold increase in percent freezing with contextual fear compared with KO mice fed the control diet. These findings indicate that response to contextual fear was improved in WT mice that consumed POP-SOS but aggravated in KO mice that consumed fish oils. Furthermore, KO mice showed a 0.4-fold decrease in percent freezing in response to tone fear when they were fed POP-SOS diet compared to a control diet. Thus, POP-SOS diet reduced tone fear level of KO mice until the same level of WT mice. Finally, KO mice fed the soybean oil diet showed a 1.7-fold increase in immobility in the forced swim test compared to KO mice fed the control diet. Taken together, oil-rich diets differentially modulate anxiety and depression in normal and anxious mice. Oils rich in saturated fatty acids may alleviate anxiety more strongly than other oils.     

Strand directionality affects cation binding and movement within tetramolecular G-quadruplexes

Nuclear magnetic resonance study of G-quadruplex structures formed by d(TG₃T) and its modified analogs containing a 5′-5′ or 3′-3′ inversion of polarity sites, namely d(3′TG5′-5′G₂T3′), d(3′T5′-5′G₃T3′) and d(5′TG3′-3′G₂T5’) demonstrates formation of G-quadruplex structures with tetrameric topology and distinct cation-binding preferences. All oligonucleotides are able to form quadruplex structures with two binding sites, although the modified oligonucleotides also form, in variable amounts, quadruplex structures with only one bound cation. The inter-quartet cavities at the inversion of polarity sites bind ammonium ions less tightly than a naturally occurring 5′-3′ backbone. Exchange of ¹⁵ ions between G-quadruplex and bulk solution is faster at the 3′-end in comparison to the 5′-end. In addition to strand directionality, cation movement is influenced by formation of an all-syn G-quartet. Formation of such quartet has been observed also for the parent d(TG₃T) that besides the canonical quadruplex with only all-anti G-quartets, forms a tetramolecular parallel quadruplex containing one all-syn G-quartet, never observed before in unmodified quadruplex structures.     

Allelopathy in black walnut (Juglans nigraL.) alley cropping. II. Effects of juglone on hydroponically grown corn (Zea maysL.) and soybean (Glycine maxL. Merr.) growth and physiology

We conducted an experiment to investigate the effects of juglone (5-hydroxy-1, 4-napthoquinone) on the growth and physiology of hydroponically grown corn (Zea mays L.) and soybean (Glycine max L. Merr.) seedlings. Three different concentrations of juglone (10^-6 M, 10^-5 M, and 10^-4 M) along with a control were applied. Within 3 days, juglone exhibited significant inhibitory effects on all measured variables including shoot and root relative growth rates (RGR_s and RGR_r), leaf photosynthesis (P_net), transpiration (E), stomatal conductance (g_s), and leaf and root respiration. In general, soybean was found to be more sensitive to juglone than corn. RGR_r was the most inhibited variable for both species, and reductions of 86.5 and 99% were observed in corn and soybean, respectively, with 10_-4 M juglone concentrations. Among the physiological variables measured, P_net showed the greatest impact of toxicity though the other physiological parameters were also impacted. We conclude that both corn and soybean are sensitive to juglone and observed growth reductions in corn and soybean in black walnut alley cropping may partly be due to juglone phytotoxicity. Determination of actual phytotoxicity will require quantification of soil solution juglone levels, particularly in areas where soil solid-phase levels are high in close proximity to trees.     

An enumerative assay of purine analogue resistant lymphocytes in women heterozygous for the Lesch-Nyhan mutation

In females heterozygous for the Lesch-Nyhan (LN) mutation, there is mosaicism of peripheral blood lymphocytes (PBLs) with regard to sensitivity to 6-thioguanine (TG) inhibition of tritiated thymidine ([³H]Tdr) incorporation following phytohemagglutinin (PHA) stimulation. That there are two populations of PBLs, normal and mutant (LN-like), has been demonstrated by an autoradiographic enumerative assay. A single three-generation family containing six potentially heterozygous females was studied. Five of the six were mosaics with frequencies of TG-resistant (TG^r) PBLs ranging from 1.4×10^–3 to 4.2×10^–3 when tested at 2×10^–4 m TG. The median frequency of TG^r PBLs in 63 healthy non-LN individuals between the ages of 11 and 75 years was found to be 1.1×10^–4 (mean 1.3×10^–4) (10th and 90th percentiles—6.1×10^–5 and 2.1×10^–4) and was not age related. The sixth potentially heterozygous female in the current family had a TG^r PBL frequency of 1.9×10^–4. In the five females with elevated TG^r PBL frequencies, TG^r skin fibroblasts with frequencies ranging from 26% to 100% of the sample tested were found; in the female with the normal TG^r PBL frequency, no TG^r skin fibroblasts were found. The former group was considered to be LN heterozygous. Four of the five had been previously diagnosed as such. The latter individual is considered to be genotypically normal. Females who are heterozygotes for the LN mutation have two populations of PBLs.This work was supported by National Institutes of Health Grant No. PHS RO1 15450 and the Surgical Associates Foundation, Inc., University of Vermont College of Medicine. Elizabeth F. Allen was supported by the NIH National Research Service Award No. 5 F32 CA 05918.     

A comparative study of myoglobin stability in the presence of Hofmeister anions of ionic liquids and ionic salts

To reveal the impact of ionic liquids (ILs) on the stability of proteins, a series of ILs possessing same 1-butyl-3-methylimidazolium cation [Bmim]⁺ with a set of Hofmeister anions such as SCN⁻, HSO₄⁻, Cl⁻, Br⁻, CH₃COO⁻ and I⁻ were used and their effects on the myoglobin (Mb) structure and stability were studied. For the sake of comparison and also to explore the extent of the stabilization behavior of ILs toward Mb stability, we have chosen a set of ionic salts (I_s) of a fixed sodium cation (Na⁺) with the same series of anions such as SCN⁻, SO₄⁻², Cl⁻, Br⁻, CH₃COO⁻ and I⁻. UV–vis, fluorescence and circular dichroism (CD) spectroscopic techniques were used in order to investigate the stability behavior of Mb in ionic species (I_s and ILs). The results reveal that both I_s and ILs had a negative influence on the stability of Mb. Apparently, the flexibility in the native structure of Mb gradually increases with the increase in the concentration of I_s and ILs at pH 7.0. Therefore, a sharp decrease in the transition temperature (T_m) of the native Mb is observed in the presence of I_s and ILs.     

Carbon source pulsed feeding to attain high yield and high productivity in poly(3-hydroxybutyrate) (PHB) production from soybean oil using <Emphasis Type="Italic">Cupriavidus necator</Emphasis>

Poly(3-hydroxybutyrate) (PHB) biosynthesis from soybean oil by Cupriavidus necator was studied using a bench scale bioreactor. The highest cell concentration (83 g l⁻¹) was achieved using soybean oil at 40 g l⁻¹ and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5 g l⁻¹ h⁻¹, and the calculated Y_p/s value was 0.85 g g⁻¹. Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe.     

Structural characterization of triacylglycerol in several oils containing gamma-linolenic acid

The differences are reported in the triacylglycerol (TG) structures of oils containing gamma-linolenic acid (GLA) from Oenothera biennis Linn seed oil (OBLO) from the wild plant, evening primrose seed oil (EPO) from a cultured plant, and bio-GLA oil (BIO) from a mold, the physiological functions of which were ascertained by animal testing. Reverse-phase high-performance liquid chromatographic separation detected 12 TG peaks each for OBLO and EPO, and 28 TG peaks for BIO. TG-containing GLA were composed of five molecular species each in OBLO and EPO, and ten molecular species in BIO. The totals of the molecular species containing GLA were 29.8% in OBLO, 23.8% in EPO, and 56.6% in BIO. In OBLO, the GLA level at the sn-2 position of the major TG species was higher than that in EPO. In BIO, the GLA level at the sn-2 position of the major TG species was lower than those in OBLO and EPO.