An energy-minimized casein submicelle working model

To develop a molecular basis for structure-function relationships of the complex milk protein system, an energy-minimized, three-dimensional model of a casein submicelle was constructed consisting of-casein, four _s1-casein, and four-casein molecules. The models for the individual caseins were from previously reported energy-minimized, three-dimensional structures. Docking of one-casein and four _s1-casein molecules produced a framework structure through the interaction of two hydrophobic antiparallel sheets of-casein with two small hydrophobic antiparallel sheets (residue 163–174) of two preformed _s1-casein dimers. The resulting structure is approximately spherically symmetric, with a loose packing density; its external portion is composed of the hydrophilic domains of the four _s1-caseins, while the central portion contains two hydrophbic cavities on either side of the-casein central structure. Symmetric and asymmetric preformed dimers of-casein formed from the interactions of C-terminal-spiral regions as a hinge point could easily be docked into each of the two central cavities of the- framework. This yielded two plausible energy-minimized, three-dimensional structures for submicellar casein, one with two symmetric-casein dimers and one with two asymmetric dimers. These refined submicellar structures are in good agreement with biochemical, chemical, and solution structural information available for submicellar casein.Reference to a brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Physiological relationship between the temperate phages lambda and phi80

Summary This work deals with the ability of phage 80 to provide defective mutants of with their missing functions. Functions Involved in Recombination. As shown by others, the Int mechanism of 80 cannot excise prophage . However, 80 efficiently excises recombinants from tandem dilysogens, using its Ter mechanism. Likewise, the nonspecific mechanism Red is interchangeable between 80 and . Maturation of DNA by 80. The Ter recombinants excised by 80 from tandem dilysogens are packaged into a 80 protein coat. This contrasts with the fact, already mentionned by Dove, that 80 is extremely inefficient for packaging phage superinfecting a -lysogen. The latter result is also found when the helper phage is a hybrid with the left arm of (80hy4 or 80hy41 — see Fig. 1). However, the maturation of the superinfecting is much more efficient if the 80hy used as a helper has the att-N region of (like 80hy1). Conversely a with the att-N region of 80 (hy6 — see Fig. 1) is packaged more efficiently by 80 or 80hy4 than by 80hy1. It is suggested that the maturation of chromosome superinfecting an immune cell requires a recombination with the helper phage. Vegetative Functions. Among the replicative functoons O and P, the latter only can be supplied by 80. That N mutants are efficiently helped by 80 does not tell that 80 provides the defective with an active N product; the chromosomes are simply packaged into a 80 coat. This shows that 80 is unable to switch on the late genes of . That neither 80 nor any of the 80hy tested can provide an active N product is shown in a more direct way by their complete failure to help N ^-r₁₄; this phage carries a polar mutation which makes the expression of genes O and P entirely N-dependant. The maturation of a N ^- by 80 contrasts with the fact that mutants affected in late genes (A, F or H) are not efficiently helped by 80. This suggests that the products coded by these genes are not interchangeable between 80 and , and that packaging of DNA into 80 coats is possible but inhibited when late proteins are present in the cell. Activation of the Late Genes. Among the im ₈₀ h ⁺ hybrids tested, only 80hy41 is able to switch on the late genes of a N defective mutant. This hybrid differs from the other hybrids studied here, by the fact that it has the Q-S-R region of (see Fig. 1). The results are consistant with the view that the product of Q gene is sufficient for activating the late genes of a DNA. N would thus control the expression of late genes only indirectly by controlling the expression of gene Q (Couturier & Dambly have independantly reached the same conclusion, 1970). Furthermore the failure of 80 and of the 80hy1 and 80hy4 to activate the late genes of would imply that these phages are unable to provide an Q product active on the chromosome Reciprocally, switches on the late genes of prophage 80hy41, but not of prophages 80hy1 and 80hy4. This suggests that the initiation of late genes expression takes place at a main specific site located in the Q-S-R region of the chromosome. The expression of the late genes would thus be sequential, and proceed through the left arm only when steaky ends cohere. Similar conclusions were reached independantly by Toussaint (1969) and by Herskowitz and Signer (1970).

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Ce travail a été réalisé dans le cadre du contrat d'association Euratom-U. L. B. 007-61-10 ABIB et avec l'aide du Fonds de la Recherche Fondamentale Collective.     

Conformational transmission in ATP synthase during catalysis: Search for large structural changes

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Defective mutation at Gly-149 was suppressed by the second mutations at the outer surface of the subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission. Extensive mutant/pseudorevertant studies revealed that / and / subunits interactions are important for the energy coupling between catalysis and H⁺ translocation. In addition, long range interaction between amino and carboxyl terminal regions of the subunit has a critical role(s) for energy coupling. These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.     

The microbial production of 3aα-H-4α- (3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-indan-1-one-δ-lactone from cholesterol

Summary A mutant strain of Rhodococcus australis CSIR-236.457 which accumulates 3a-H-4-(3-propionic acid)-5-hydroxy-7a-methylhexahydro-indan-1-one--lactone from cholesterol, stigmasterol and -sitosterol was studied. The product is produced in a 60% molar yield in a dilute black strap molasses medium containing 6–12g/l cholesterol after a 72 hour fermentation period.     

First controlled progenies checked by isozymic markers in cultivated yams Dioscorea cayenensis-rotundata

As tested progeny have never been obtained, breeding studies on African yams (Dioscorea cayenensisrotundata) are scarce. We report here the first progenies checked by isoenzyme markers. This was made possible by the choice of well-known genitors [one male (cv Zrezrou) and three females (cvs Sopéré, Dahomey and C 20)] and special hybridization conditions. Six enzymatic systems [esterase (EST), isocitrate dehydrogenase (ICD), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6PGD), shikimate dehydrogenase (SDH), and phosphoglucoisomerase (PGI)] were used to check the progenies and detect outbreeding. Despite the small number of progeny, it was possible to provide information on the genetics of the isoenzymatic systems.     

Production of Recombinant Endostatin in Milk of Transgenic Mice

Using the bovine S1-casein gene, a genetic construct with an endostatin-coding fragment of the mouse collagen XVIII cDNA was designed to express endostatin in milk of transgenic animals. Several transgenic mice were obtained. The mice secreted endostatin in milk at 70–300 ng/l and transmitted this character to their progeny.     

Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture

Embryogenesis from isolated microspore cultures of wheat was improved by ovary co-culture when compared to a completely defined medium. This indicates that essential factor(s) in addition to PAA or its analogs may be supplied by the ovaries. Isolated microspores cocultured with 20 ovaries of wheat on the top of semi-solid MMS3 induction medium for 21–30 days gave the best response. Both the number and quality of the embryos was significantly increased. The maximum frequencies of dividing microspores and of embryogenesis were 94% and 2.4%, respectively. Up to 2583 embryos were formed per 100 anthers of cv Chris and between 18% and 43% of the larger embryos regenerated into green plants upon transfer. Genotype differences for both induction and embryogenesis phases were reduced using ovary co-culture. However, there was still a strong genotype influence on plant regeneration with cv Chris, with the F1 of Chris × Sinton displaying the highest frequencies. These results are important with respect to enhancing haploidy applications in wheat biotechnology and plant breeding.Abbreviations PAA Phenylacetic acid - MMS modified MS medium - MS Murashige and Skoog's medium 1962 - FHG Hunter's FHG medium 1988     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1 (EF-1), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1. An intron is located in the 5 untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1tef-c (97%) than to the P. purpurea EF-1tef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.     

Partitioning of water resources among plants of a lowland tropical forest

Source water used by plants of several species in a semi-evergreen lowland tropical forest on Barro Colorado Island, Panama, was assessed by comparing the relative abundance of deuterium, D, versus hydrogen, H (stable hydrogen isotope composition, D) in xylem sap and in soil water at different depths, during the dry season of 1992. Ecological correlates of source water were examined by comparing xylem water D values with leaf phenology, leaf water status determined with a pressure chamber, and rates of water use determined as mass flow of sap using the stem heat balance method. Soil water D values decreased sharply to 30 cm, then remained relatively constant with increasing depth. Average D values were-13, for 0–30 cm depth and-36.7 for 30–100 cm depth. Soil water D values were negatively associated with soil water content and soil water potential. Concurrent analyses of xylem water revealed a high degree of partitioning of water resources among species of this tropical forest. Xylem water D of deciduous trees (average=-25.3±1.4) was higher than that of evergreen trees (average=-36.3±3.5), indicating that evergreen species had access to the more abundant soil water at greater depth than deciduous species. In evergreen shade-tolerant and high-light requiring shrubs and small trees, D of xylem water was negatively correlated with transpiration rate and leaf water potential indicating that species using deeper, more abundant water resources had both higher rates of water use and more favorable leaf water status.     

Heterogeneity of epithelial cells in the human thymus

Summary To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: subcapsular-perivascular (type 1); pale (type 2); intermediate (type 3); dark (type 4); undifferentiated (type 5); and large-medullary (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm).Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction (thymic nurse cells), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.     

Purification and immunocytochemical localization of kafirins inSorghum bicolor (L. Moench) endosperm

Summary Kafirins are the storage proteins of sorghum and are found in protein bodies in the seed endosperm. They have been classified as -, -, and -kafirins according to differences in molecular weight, solubility, and structure. The kafirins were purified, amino acid composition was determined, and immunolocalization methods were used to determine the organization of the protein bodies and distribution of kafirins throughout the endosperm. All three groups of kafirins were low in lysine. -Kafirins and -kafirins were relatively high in cysteine, and -kafirins were relatively high in methionine. Transmission electron microscopy showed that protein bodies in the peripheral endosperm were spheroid with concentric rings and few darkly stained inclusions. In contrast, protein bodies of the central endosperm were irregularly shaped with a higher proportion of darkly stained material. The light staining regions of the protein bodies are composed primarily of -kafirins with minor portions of - and -kafirins. The dark staining regions, however, are composed primarily of - and -kafirins. Immunoelectron microscopy showed that protein bodies in the peripheral endosperm contain predominantly a-kafirin with minor amounts of - and -kafirin. Central endosperm protein bodies are also predominantly -kafirin, but have a higher proportion of -kafirin and -kafirin than the peripheral endosperm protein bodies.Abbreviations GAR-HRP Goat anti-rabbit horseradish peroxidase - IgG immunoglobulin G - 2-ME 2-mercaptoethanol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffer saline - TBS-T Tris buffer saline with Tween - TBS-T-B Tris buffer saline with Tween and bovine serum albumin - TCA trichloroacetic acid - UV ultraviolet     

Chemical synthesis,expression and mutagenesis of a gene encoding β-cryptogein,an elicitin produced by Phytophthora cryptogea

Elicitins are 10 kDa holoproteins secreted by Phytophthora fungi, that elicit an incompatible hypersensitive reaction, leading to resistance against fungal and bacterial plant pathogens. Comparison of primary sequences of -elicitins and -elicitins indicated several potential necrotic activity-determining residues. All of the highly necrotic -elicitins have a hydrophilic residue (usually lysine) at position 13, whereas in the less necrotic -elicitins this residue is replaced by a valine. Here, we report the synthesis and expression of a gene encoding a highly necrotic elicitin, -cryptogein, and we show that the substitution of Lys-13 of this recombinant protein by a valine leads to a drastic alteration to the necrotic activity of the recombinant protein.     

SDS Induced Structural Changes in α-Crystallin and It’s Effect on Refolding

-Crystallin, a major eye lens protein and a key member of the small heat shock protein family, acts like a chaperone by preventing aggregation of substrate proteins. One of the hallmarks of most small heat shock proteins is their existence as a large oligomer, the role of which in its function is not understood at present. We have studied the role of the oligomer in the stability of its structure against SDS induced destabilization by CD measurements. -Crystallin from bovine source as well as recombinant preparation was used for this purpose. As SDS concentration was gradually increased, the -sheet structure was diminished followed by concomitant increase in the -helical structure. The quaternary structural changes in presence of SDS were also monitored by light scattering, polarization and anisotropy measurements. It was found that the breakdown of the oligomeric structure was nearly complete above 1 mM SDS concentration. The results were compared with that of a monomeric -crystallin, which is also a major -sheet protein like -crystallin. When -crystallin was first converted into monomeric random coil structure in presence of 6 M urea and allowed to refold in SDS solution, amount of -helix was more than that incubated directly in the same concentration of SDS. The results show that -crystallin attains extra structural stability against external stress due to its oligomeric structure. The implication for the extra stability is discussed in reference to its function as molecular chaperone.     

Alpha-tocopherol in the retinal outer segment of bovine eyes

Summary -Tocopherol was identified in lipid extracts of bovine retinal outer segment (ROS) preparations. Positive identification was obtained by the thin layer chromatographic characteristics of the tocopherol form and its oxidation product -tocopherylquinone, and by the ultraviolet spectrum of the oxidized and KBH₄-reduced form of the tocopherylquinone. In the ROS preparations used, -tocopherol chromanol was the predominant species, the quinone form accounting for 25% or less of the total. The concentration of -tocopherol in the ROS preparations was about 0.1 mole -tocopherol per mole rhodopsin, or about 1 nmole/mg, protein. Mitochondria from bovine retina contained about 0.4 nmole -tocopherol per mg protein.     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Effects of exercise training and diabetes on cardiac myosin heavy chain composition

This study determined whether the beneficial effects of exercise training on the diabetic heart previously observed are associated with alterations in ventricular myosin heavy chain (MHC) isoform composition. Diabetes was induced in rats by i.v. streptozotocin. Trained rats were run on a treadmill for 60 min/day, 27 m/min, 10% grade. After 10 wks, ventricular MHC isoenzyme protein composition was analyzed for MHC composition using gel electrophoresis. -MHC and -MHC mRNA were determined by Northern and slot blot hybridization techniques. Both protein and mRNA analyses indicated that sedentary control rats exhibited a predominance of -MHC. Sedentary diabetics exhibited a shift to -MHC. Exercise trained diabetic rats showed a predominance of -MHC. The results indicate that treadmill exercise training of diabetic rat does not prevent the diabetes-induced shift in MHC composition towards the -MHC isoform, thus it is unlikely that the beneficial effects of exercise training on the diabetic heart, previously shown, are due to a normalization of the myosin isoform composition.     

Production of pineapple plants in vitro

In vitro culture of pineapple (Ananas comosus) was studied to determine the efficiency of axillary bud culture for rapid propagation of several cultivars. The technique used maximizes the success rate of various steps in the production of pineapple plants. Rapid mass multiplication of plantlets started 9 months after explanting with a significant log phase. The number of plantlets obtained from the culture of a single bud by the thirteenth month ranged from 210 to 380 for Perolera; 300 to 350 for PR-1-67; and 40 to 85 for Smooth Cayenne. The method permits culture of a range of pineapple cultivars. Little morphological variation was observed in young regenerated plants.     

Concluding remarks

On the basis of symposium contributions onChlorella, Hibbertia, Eucalyptus, Ambrosia and on numerical approaches some fundamental problems of (bio)systematics, evolution, and taxonomic categories are discussed: Methods available for analysing affinities; conflicting evidence from phenetic, biochemical, cytogenetic and other analyses; further classification problems in cases of intermediacy, etc. While sibs of various levels and their natural hierarchy often can be objectively defined, this appears impossible for particular taxonomic levels itself (e. g. species). A single objective taxonomic system of organisms is unrealistic. Certain guiding lines for relative and practicable concepts of species and genus are proposed.Presented at the symposium Speciation and the Species Concept during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

The influence of increasing chlorine content on the accumulation and metabolism of polychlorinated biphenyls (PCBs) by Paul's Scarlet Rose cells

Four radiolabled congeners of biphenyls with increasing chlorine content (biphenyl; 1-monochlorobiphenyl; 2,2,4,4-tetrachlorobiphenyl; and 2,2,4,4,5,5-hexachlorobiphenyl) were provided to suspension cultures of rose (Rosa sp. cv. Paul's Scarlet) for 4 days. Both the kinetics of ¹⁴C exchange between the cells and medium, and the metabolism of the parent compounds depended on the chlorine content of the congeners. Analysis of both the cells and their medium showed that of the recovered radioactivity 88%, 86%, and 3% of the biphenyl, 1-PCB, and 2,2,4,4-PCB were metabolized respectively to polar and insoluble residue products. The 2,2,4,4,5,5-PCB did not appear to be metabolized.