State and time delay estimation of continuous microorganisms cultivation

A continuous fermentation model taking into account the culture memory is used for a state estimation design. The influence of the culture memory on the process dynamics is accounted for by a time delay parameter. The proposed procedure of on-line state estimation in the case when the delay has a constant value is based on the extended Kalman observer. The case when the delay parameter is evaluated on-line is also considered. An adaptive state and parameter algorithm on the base of the extended Kalman filter is proposed. The theoretical results are applied to continuous culture for growth of a strain of Saccharomyces cerevisiae.List of Symbols X, S mg/l Biomass concentration and substrate concentration respectively - S ⁰ mg/l Feed substrate concentration - Z mg/l Past substrate concentration - µ h^–1 Specific growth rate taking into account culture memory - h^–1 Specific consumption rate - h Time delay parameter denoting culture memory - D h^–1 Dilution rate - State variables vector - W _ij Gain coefficient for on-line state and parameter estimation - F Substrate feed rate vector - () Gain coefficient matrix - R Square symmetric Riccati matrix - K Matrix of coefficients - K(t) Delay kernel taking account of culture memory - Denote an estimation value The partial support by Bulgarian National Science Research Foundation under Grant SRTS 428/94 Modeling and Control of Fermentation Processes Taking the Memory Effect into Account is gratefully acknowledged.     

Genetic analysis of resistance to whitebacked planthopper in twenty-one varieties of rice,Oryza sativa L.

Summary The inheritance of resistance to whitebacked planthopper Sogatella furcifera (Horvath) was studied in 21 rice varieties. Reactions of F₁; F₂ and F₃ progenies of the crosses of 21 resistant varieties with the susceptible variety TN 1 revealed that a single dominant gene governs resistance in Mushkan 41, Santhi, Siahnakidar 195, SM2-34, Tirisurkh 251, Zirijowaian 245, 18, 24A, 39, 76 S, 78, 180, 213 B, 267, 293, CI 6037-4, NP97, S39 JKW and Bansphul. In varieties 65 and 274 A, resistance is governed by one dominant and one recessive gene which segregate independently of each other. Tests for allelism with the Wbph 1 gene originally identified in N 22 revealed that the dominant gene present in all the test varieties is the same as Wbph 1. Further studies are required to determine the allelic relationships of the recessive gene found in varieties 65 and 274 A.     

Disentangling a rainforest food web using stable isotopes: dietary diversity in a species-rich ant community

For diverse communities of omnivorous insects such as ants, the extent of direct consumption of plant-derived resources vs. predation is largely unknown. However, determination of the extent of "herbivory" among ants may be crucial to understand the hyper-dominance of ants in tropical tree crowns, where prey organisms tend to occur scarcely and unpredictably. We therefore examined N and C stable isotope ratios (¹⁵N and ¹³C) in 50 ant species and associated insects and plants from a tropical rainforest in North Queensland, Australia. Variation between ant species was pronounced (range of species means: 7.1 in ¹⁵N and 6.8 in ¹³C). Isotope signatures of the entire ant community overlapped with those of several herbivorous as well as predacious arthropods. Variability in ¹⁵N between ants was not correlated with plant ¹⁵N from which they were collected. Ant species spread out in a continuum between largely herbivorous and purely predacious taxa, with a high degree of omnivory. Ant species' ¹⁵N were consistent with the trophic level predicted by natural feeding observations, but not their ¹³C. Low ¹⁵N levels were recorded for ant species that commonly forage for nectar on understorey or canopy plants, intermediate levels for species with large colonies that were highly abundant on nectar and honeydew sources and were predacious, and the highest levels for predominantly predatory ground-foraging species. Colonies of the dominant weaver-ants ( Oecophylla smaragdina) had significantly lower ¹⁵N in mature forests (where preferred honeydew and nectar sources are abundant) than in open secondary vegetation. N concentration of ant dry mass showed only very limited variability across species and no correlation with trophic levels. This study demonstrates that stable isotopes provide a powerful tool for quantitative analyses of trophic niche partitioning and plasticity in complex and diverse tropical omnivore communities.     

Spatiotemporal expression patterns of sialoglycoconjugates during nephron morphogenesis and their regional and cell type-specific distribution in adult rat kidney

The expression of 2,6- and 2,3-linked sialic acids on N-glycans was studied in embryonic, postnatal, and adult rat kidney. Histochemistry and blotting using Polyporus squamosus and Sambucus nigra lectins for 2,6-linked sialic acids and the Maackia amurensis lectin for 2,3-linked sialic acids were performed and sialyltransferase activity was assayed. N-glycans with 2,6- and 2,3-linked sialic acid were differently expressed in the two embryonic anlagen and early stages of nephron. Metanephrogenic mesenchyme was positive for 2,3-linked sialic acid but not for the 2,6-linked one, which became detectable initially in the proximal part of S-shaped bodies. Collecting ducts were positive for 2,6-linked sialic acid, whereas 2,3-linked sialic acid was restricted to their ampullae. Although positive in embryonic kidney, S1 and S2 of proximal tubules became unreactive for 2,3-linked sialic acid in postnatal and adult kidneys. In adult kidney, intercalated but not principal cells of collecting ducts were reactive for 2,3-linked sialic acid. In contrast, 2,6-linked sialic acids were detected in all cells of adult kidney nephron. Blot analysis revealed a different but steady pattern of bands reactive for 2,6- and 2,3-linked sialic acid in embryonic, postnatal, and adult kidney. Activity of 2,6 and 2,3 sialyltransferases was highest in embryonic kidney and decreased over postnatal to adult kidney with the activity of 2,6 sialyltransferase always being three to fourfold that of 2,3 sialyltransferase. Thus, 2,6- and 2,3-linked sialic acids are differently expressed in embryonic anlagen and mesenchyme-derived early stages of nephron and show regional and cell type-specific differences in adult kidney.     

Identification of functionally important cysteines in the alpha-subunit of transducin by chemical cross-linking techniques

Transducin (T), the G-protein in the visual system, is a heterotrimer arranged as two functional units, T and T. N, N-1, 2-phenylenedimaleimide (o-PDM) and N, N-1, 4-phenylenedimaleimide (p-PDM), two cysteine specific-homobifunctional agents, were used to covalently cross-link T and its units. A complete inhibition in T function was observed in the presence of these compounds. Incubation of T with o-PDM or p-PDM resulted in the formation of high-molecular-weight oligomers of 70-, 105-, 140-, and 200 kDa, as well as intramolecular cross-linked polypeptides that migrated as 35- and 37-kDa bands. Additionally, the treatment of T with both reagents produced a major species of 46-kDa. The combination of intact T and o-PDM- or p-PDM-treated T reconstituted T native activities. On the contrary, when o-PDM- or p-PDM-modified T was incubated with intact T, more than 90% inhibition on T function was observed. Hence, the cysteines modified and/or cross-linked on T represent functionally important residues of T.     

Cytoplasmic gel and water relations of axon

Summary A previous method of measuring the swelling pressure ( _g ) of the cytoplasmic gel of the giant axon ofLoligo vulgaris was refined. The estimates of _g made with the improved method were consistent with those made with the earlier method. In these methods the activity of the solvent in the gel is measured by increasing the activity of the solvent in the internal phase of the gel by application of hydrostatic pressure to the gel directly. Comparable values for the activity of the solvent in the gel were obtained also by an alternate method, in which the deswelling of the gel is measured upon decreasing the activity of the solvent in the external phase by addition of a nonpenetrating high mol wt polymer (i.e., Ficoll).Additional support was obtained for the earlier suggestion that _g contributes to the swelling and shrinkage pattern of the whole axon. In part, the new evidence involved two consecutivedirect measurements of intraxonal pressure. The first measurement was that of a mixed pressure composed of _g and _m ( _m being the effective osmotic pressure due to the intra-extraxonal gradient in the activity of mobile solutes). The subsequent measurement was that of _g alone. The latter measurement was made feasible by destroying the axolemma, thereby eliminating the contribution of _m . An estimate of _m was obtained by subtracting _g from the total pressure measured initially. The _m determined by the above method was two orders of magnitude smaller than the theoretical osmotic pressure. This is consistent with the _m determined previously, where osmotic intra-extraxonal filtration coefficients were compared to the hydrostatic. The mixed pressure experiments lend credence to the idea that the substantial contribution of _g to the water relations of the whole axon is due to _g being of the same order of magnitude as _m .The degree of free swelling of axoplasmic gels was studied as a function of pH, salt concentration, and hydration radius of the anion of the salt used. The swelling increased with an increase in the reciprocal of the hydration radius, a decrease in salt concentration, and at pH below or above 4.5.The nature of the constraints to the free swelling of axoplasm in axons immersed in seawater was studied. With the seawater employed, these constraints appeared to be due more to the retractive forces of the sheath than to _m .     

The mucin profiles of normal gastric mucosa, intestinal metaplasia and its variants and gastric carcinoma

Summary Human gastrectomy specimens, including 48 carcinomas and 25 selected benign cases showing extensive intestinal metaplasia (IM), were examined in detail histologically and histochemically. IM was classified into complete (type I), incomplete without sulphomucins (type IIA) and incomplete with sulphomucins (type IIB). Type IIB was associated with intestinal but not diffuse cancers (P<0.01) or benign lesions (P<0.01). The mucin profiles of IIB IM were similar to intestinal cancers with sulphomucins predominating overN-acetylsialomucins.O-acetylsialomucins were not seen in IIB IM or tumours but were observed in complete or type IIM. These findings suggest a histogenic link between incomplete IM secreting sulphomucins (or colonic metaplasia) and intestinal cancers of the human stomach.     

An ischemic β-dystroglycan (βDG) degradation: Correlation with irreversible injuiry in adult rabbit cardiomyocytes

A loss of sarcolemmal dystrophin was observed by immuno-fluorescence studies in rabbit hearts subjected to in situ myocardial ischemia and by immuno-blotting of the Triton soluble membrane fraction of isolated rabbit cardiomyocytes subjected to in vitro ischemia. This ischemic loss of dystrophin was a specific event in that no ischemic loss of sarcolemmal -sarcoglycan, -sarcoglycan, DG, or DG was observed. The maintenance of sarcolemmal DG (43 Kd) during ischemia was interesting in that dystrophin binds to the C-terminus of DG. However, during late in vitro ischemia, a 30 Kd band was observed that was immuno-reactive for DG. Additionally, this 30 Kd-DG band was observed in rabbit myocardium subjected to autolysis. Finally, the 30 Kd-DG was observed in the purified sarcolemmal fraction of rabbit cardiomyocytes subjected to a prolonged period of in vitro ischemia, confirming the sarcolemmal localization of this band. The potential patho-physiologic significance of this band was indicated by the appearance of this band at 120–180 min of in vitro ischemia, directly correlating with the onset of irreversible injury, as manifested by osmotic fragility. Additionally the appearance of this band was significantly reduced by the endogenous cardioprotective mechanism, in vitro ischemic preconditioning, which delays the onset of osmotic fragility. In addition to dystrophin, DG binds caveolin-3 and Grb-2 at its C-terminus. The presence of Grb-2 and caveolin-3 in the membrane fractions of oxygenated and ischemic cardiomyocytes was determined by Western blotting. An increase in the level of membrane Grb-2 and caveolin-3 was observed following ischemic preconditioning as compared to control cells. The formation of this 30 Kd-DG degradation product is potentially related to the transition from the reversible to the irreversible phase of myocardial ischemic cell injury and a decrease in 30 Kd-DG might mediate the cardioprotection provided by ischemic preconditioning.     

Regulation of isopenicillin N synthetase and deacetoxycephalosporin C synthetase by carbon source during the fermentation of Cephalosporium acremonium

Summary In a chemically defined medium with glucose and sucrose as major carbon sources (standard medium), Cephalosporium acremonium excretes the intermediate of the -lactam biosynthetic pathway, penicillin N, into the medium during growth; production of cephalosporins is delayed until glucose is completely utilized. Deacetoxycephalosporin C synthetase, the ring-expansion enzyme (expandase), does not appear as long as glucose is present. Afterwards, initiation of its formation is accompanied by the production of cephalosporins. Feeding additional glucose during the fermentation turns off expandase synthesis without affecting formation of isopenicillin N synthetase, the ring-cyclization enzyme (cyclase). The above results point to a strong glucose catabolite repression of the expandase as one of the main regulatory mechanisms in -lactam biosynthesis by Cephalosporium acremonium and the reason for accumulation of penicillin N during the fermentation. Cyclase shows a biphasic pattern in activity, the first very high peak not being correlated with the excretion of any -lactam antibiotic into the medium.     

The composition of Erythrosins, Fluorescein, Phloxine and Rose Bengal: a study using thin-layer chromatography and solvent extraction

Synopsis Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4-iodofluorescein, 4,5-di-iodofluorescein, 2,4,5-triiodofluorescein and 2,4,5,7-tetraiodofluorescein, in some instances together with 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4-bromo-4,5,6,7-tetrachlorofluorescein, 4,5-dibromo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tribromo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4-iodo-4,5,6,7-tetrachlorofluorescein, 4,5-di-iodo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.     

Nutrient proportions in foliage of semi-mature loblolly pine

Summary Nitrogen and phosphorus limitations to growth are common in many loblolly pine (Pinus taeda) stands. Interactions of these nutrients may complicate interpretation of foliar nutrient analysis for predicting response to forest fertilization. Proportions of foliar nutrient concentrations (and the changes in these proportions following fertilization) were examined in 36 semi-mature loblolly pine plantations in the southeastern United States. Mean proportions of nutrient concentrations (NPKCaMg) for non-fertilized stands were 1009.336.517.29.2. Potassium and phosphorus were higher. Nitrogen fertilization generally decreased the PN ratio and enhanced growth, indicating a nitrogen deficiency in most stands under study. Additions of nitrogen and phosphorus together yielded a significant increase in the PN ratio. Effects of fertilization effects on other nutrient concentration ratios were also examined.Paper No. 9401 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes

The structures of gangliosides from human granulocytes were elucidated by fast atom bombardment mass spectrometry and by gas chromatography/mass spectrometry as their partially methylated alditol acetates. In human granulocytes besides G_M3 (II³Neu5Ac-LacCer), neolacto-series gangliosides (IV³Neu5Ac-nLcOse₄Cer, IV⁶Neu5Ac-nLcOse₄Cer and VI³Neu5Ac-nLcOse₆Cer) containing C_24:1, and to some extent C_22:0; and C_16:0 fatty acid in their respective ceramide portions, were identified as major components. In this study we demonstrate that gangliosides from human granulocytes, the second most abundant cells in peripheral blood, can serve as receptors for influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and a parainfluenza virus Sendai virus (HNF1, Z-strain). Viruses were found to exhibit specific adhesion to terminal Neu5Ac2-3Gal and/or Neu5Ac2-6Gal sequences as well as depending on the chain length of ganglioside carbohydrate backbones from human granulocytes, these important effector cells which represent the first line of defence in immunologically mediated reactions. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GC/EIMS, gas chromatography/electron impact mass spectrometry; GSL(s) glycosphingolipids; HPTLC, high performance thin-layer chromatography; Neu5Ac,N-acetylneuraminic acid [26], PFU, plaque forming unit. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations, and the ganglioside nomenclature system of Svennerholm was used. LacCer or lactosylceramide, Gal1-4Glc1-1Cer gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; lacto-N-tetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4-Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse₆Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal 1-4-Glc1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer; G_T1b, IV³Neu5Ac, II³(Neu5Ac)₂-GgOse₄Cer; G_Q1b, IV³(Neu5Ac)₂, II³(Neu5Ac)₂-GgOse₄Cer; sialyllacto-N-tetraosylceramide, IV³Neu5Ac/IV⁶Neu5Ac-nLcOse₄Cer; sialyllacto-N-norhexaosylceramide or i-active ganglioside, VI³Neu5Ac-nLcOse₆Cer.     

Isozyme analysis as a tool for introgression of Sinapis alba germ plasm into Brassica napus

Summary Isozyme analysis of Brassica napus cv Topas and CGRC5006 as well as of Sinapis alba cv Emergo revealed significant polymorphism between the two species for the isozymes, aconitate hydratase, glucose phosphate isomerase, and diaphorase. F₁ hybrids between B. napus 5006 and S. alba cv Emergo were backcrossed to B. napus cv Topas, and the S₁ progeny of the first two backcrosses were studied isozymically. At the backcross one level the frequency of S. alba or S. alba plus B. napus patterns observed ranged from 18% to 87% across the four lines studied. There were differences between lines for the frequency of S. alba patterns, which could have an impact on the efficiency of selection for subsequent backcrossing. By the backcross two generation in one of the two lines studied, GR86-24, the S. alba patterns for GPI and DIA had been lost, while in the other line, GR86-28, the S. alba pattern for ACO had been lost, resulting in lost opportunity for S. alba gene transfer. In a wide cross such as S. alba x B. napus, which requires an intensive effort to accomplish, the isozymes ACO, GPI, and DIA may serve as useful markers to ensure gene transfer between the two species has occurred. In addition, the identification of lines with divergent isozyme patterns from B. napus will provide the basis for establishing linkages between S. alba traits of interest and isozyme markers.     

Involvement of abscisic acid in bulb dormancy of Allium wakegi Araki. II. A comparison between dormant and nondormant cultivars

The content of abscisic acid (ABA) in bulbs of two Allium wakegi Araki cultivars, Kiharabansei No. 1 (dormant type) and Ginoza (nondormant type), was similar and changed similarly during the development and storage of the bulbs. It increased during bulb development, reached a maximum shortly after bulb harvesting, and gradually decreased during bulb storage. The bulbs of Kiharabansei No. 1 showed dormancy correlated with the change in ABA content, but those of Ginoza did not show significant dormancy throughout the experimental period. The ABA content in the buds of dormant bulbs of Kiharabansei No. 1 did not change after planting of bulbs, but that of nondormant bulbs of Ginoza planted on the same day rapidly decreased after planting. Application of ABA to bulbs delayed sprouting of both cultivars, but dormant bulbs of Kiharabansei No. 1 had higher sensitivity to ABA than the bulbs of Ginoza or the bulbs of Kiharabansei No. 1 partly released from dormancy. These results suggest that the decrease in the ABA content after planting (watering) and low sensitivity to ABA are correlated with the nondormancy of Ginoza.     

Sequence of a cDNA coding for a rat class II Aα chain: Extensive DNA and protein sequence identity to H-2 Aα and HLA-DC1 α chains

The rat major histocompatibility complex (RT1-B region) codes for two sets of class II molecules (la antigens) referred to as A and E. Each class II molecule is composed of two glycoprotein chains called the A and A or E and E . Two cDNA clones encoding rat A chains were identified from cDNA derived from rat spleen mRNA using a combination of mRNA selection and colony hybridization techniques. The complete nucleotide sequence of the cDNA insert of one of these cDNA clones, pRIa.2, was determined. This sequence codes for the carboxy-terminal 129 amino acids of the rat A chain and 293 nucleotides of 3 untranslated sequence. The rat A chain was shown to be highly homologous in terms of both protein and DNA sequence identities to HLA-DC and H-2 A chains. Comparison between the coding regions of the cDNA insert of pRla.2 and the corresponding region of a cDNA insert encoding an HLA-DC1 chain showed sequence identities of 85% and 81% at the protein and DNA levels, respectively. Comparison between pRIa.2 and cDNA encoding an H-2 A chain sequence showed identities of 91% for both protein and DNA. Results are discussed which strongly suggest that the class II A and E primordial genes arose by gene duplication prior to the evolutionary divergence of the mammals.     

Glycerolipid-fatty-acid desaturase deficiencies in chloroplasts from fruits of Capsicum annuum L.

Chloroplasts from fruits and leaves of Capsicum annuum cv. Bell Tower were purified on sucrose gradients, and the lipids were separated by column and thin-layer chromatography. The glycerolipids mono- and digalactosyldiacylglycerol (MGDG, DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG) were quantified, and the fatty-acid composition at the 1 and 2 positions of the glycerol moiety (sn-1 and sn-2) was determined after hydrolysis with position-specific lipases. In fruit chloroplasts, ³-trans hexadecenoate (trans-3-161) was absent and replaced by palmitate (160) at sn-2 of PG, and ^7,10,13-hexadecatrienoate (163) at sn-2 of MGDG was greatly reduced and largely replaced by linoleate (182). The ratio of 182 to linolenate (183) was consistently greater in glycerolipids from fruit compared with leaf chloroplasts. The lower percentage of C-16 fatty acids at sn-2 indicated that prokaryotic molecular species were reduced by 15% in DGDG, 40% in SQDG, and 90% in MGDG, in fruit compared with leaf chloroplasts. The MGDGDGDG ratios in fruit and leaf chloroplasts were 1.21 and 2.21, respectively. Taken together, the data indicate that chloroplasts in Capsicum fruit are deficient in three desaturases: those that convert 1) 160 to ³-trans-161 at sn-2 of PG, 2) 160 to ⁷–cis-161 at sn-2 of MGDG, and 3) 182 to 183 at both sn-1 and sn-2 of various chloroplast glycerolipids.Abbreviations Chl chlorophyll - DGDG digalactosyldiacylglycerol - FS free sterol - GL galactolipid - MGDG monogalactosyldiacylglycerol - PE phosphatidyl ethanolamine - PG phosphatidylglycerol - PL phospholipid - SQDG sulfoquinovosyldiacylglycerol We are grateful to Dr. Roger Calza for providing us with the tobacco gt11 cDNA expression library and to Dr. Eric Huttner for his advice throughout the screening procedure. We also wish to thank M. Gosse for his assistance in growing and maintaining our plants. T.W.B. was supported by a BAP research grant from the Commission of the European Communities.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.