Immunofluorescence localization of the epidermolytic toxin target in mouse epidermal cells and tissue

Summary An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a direct fluorescence procedure using FTC-toxin, but not by an indirect procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the direct and indirect procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the indirect procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a direct procedure using FTC-antiprofilaggrin but only weakly by an indirect double antibody procedure. Insensitivity to indirect procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.     

Ecological theories and Dutch nature conservation

This paper aims to achieve insight into various ecological theories in the Netherlands which have different, and sometimes opposing, views on the conservation of nature. Interviews, publications and archival research brought to light four separate theories: vitalistic/holistic, dynamic, cybernetic and chaos. Diversity is reached through stability according to vitalistic/holistic and cybernetic theories, but through change and instablility according to the dynamic and chaos theories. These two groups are working apart, and continue to have their own ideas. Prediction of the future is only possible with the vitalistic/holistic and cybernetic theories. Ecologists who adhere to these theories feel responsible and able in different ways to change ecological nature towards desirable end goals. The other two theories, dynamic and chaos, appear to be less activist.     

Identification of amino acids involved in the binding of hMIP-1α to CC-CKR1, a MIP-lα receptor found on neutrophils

Human macrophage inflammatory protein-1 (hMIP-1) and human macrophage inflammatory protein-1 (hMIP-1) are chemokines involved in a diverse range of immunological effects. Both hMIP-1 and hMIP-1 are involved in the activation of monocytes and THP-1 cells probably through a common receptor(s). However, only hMIP-1 can bind to neutrophils with high affinity, presumably through CC-CKR1 (CKR1). Since the structure of these two proteins is highly conserved, non-conserved amino acids must define the disparate binding patterns that these two proteins exhibit. Measurements of binding, chemotaxis and calcium influx conducted with hMIP-1 and hMIP-1 chimeric proteins and mutants show that two amino acids (37K and 43L) are important in the binding and signaling of hMIP-1 through CKR1. Furthermore, we also show that mutations of the three charged amino acids at the C-terminus of hMIP-1 and hMIP-1 (amino acids 61, 65 and 67), do not adversely affect the binding to THP-1 cells.     

Relationships of Agropyron intermedium chromosomes determined by chromosome pairing and alcohol dehydrogenase isozymes in common wheat background

Summary The relationships of Agropyron intermedium chromosomes in two wheat-Agropyron addition series were determined. Chromosome pairing behaviour revealed that the alien chromosome in lines TAF-2 and L7 of Vilmorin-A. intermedium set are homologous to the alien chromosomes in lines P and C of the Caribo-A. intermedium set respectively. Localization of alcohol dehydrogenase isozyme genes in Vilmorin-Agropyron addition line L4 and in Caribo-Agropyron line O indicated relationships with wheat chromosomes of homoeologous group 4.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Expression of α-amylase in response to wounding in mung bean

The activity of -amylase (EC 3.2.1.1) in mung bean (Vigna radiata (L.) Wilczek) cotyledons increased markedly in response to wounding. The changes in enzyme activity were in parallel with those in enzyme content. The level of -amylase mRNA also notably increased in wounded cotyledons and attained its maximum level during the period between 1 and 2 d after wounding. The level of mRNA for phenylalanine ammonia-lyase, which is one of the well-characterized stress-inducible proteins, also increased after wounding, but the increase in mRNA level was faster than that of -amylase mRNA. On the other hand, the content of mRNA for actin, a housekeeping protein, was almost the same in wounded and unwounded cotyledons. The increase in -amylase mRNA level in wounded cotyledons was severely inhibited by -amanitin and cordycepin. -Amylase expression in the first leaves of mung-bean seedlings was also induced by wounding.Abbreviations PAL phenylalanine ammonia-lyase - SSC standard saline citrate We greatly acknowledge Prof. Richard Meagher, Department of Genetics, University of Georgia, Athens, USA for the gift of soybean actin gene clone. We also thank Mr. Kaoru Ishiwata for technical assistance.     

Effect of starvation on the content of free amino acids in plasma of different breeds of hen

Summary Leghorn, Cornish and White Rock hens were subjected to starvation. Free amino acids were determined in blood samples taken after 48, 72 and 96 h of starvation. A progressive decrease in concentration of the majority of amino acids was found. Changes in amino acid concentrations during starvation were dependent on the breed of hen.     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Study on the vesicular-arbuscular mycorrhiza of three cultivars of potato (Solanum tuberosum L.)

Summary The establishment and development of vesicular-arbuscular mycorrhizal (VAM) fungi were studied in three cultivars of potato, which differed in susceptibility to Late blight, in a field experiment on a lateritic sandy-loam during two growing seasons (1980 and 1981). The cultivars SSC 1174 (highly resistant) and Kufri Jyoti (resistant) showed an earlier establishment and more rapid development of VAM fungi than up-to-date (highly susceptible). The first mycorrhizal infection in both SSC 1174 and Kufri Jyoti was observed after 12 days in 1980 and 8 days in 1981, whereas in up-to-date it was observed after 19 and 12 days respectively. The mycorrhizal infection increased with the age of the plants in all the three cultivars.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

3α-Hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

Summary The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3-hydroxysteroid and the 3-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6.During the entire estrous cycle, 3-hydroxysteroid dehydrogenase and 3-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.This investigation was supported by the Deutsche Forschungsgemeinschaft     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Quercetin suppresses proinflammatory cytokines production through MAP kinases and NF-κB pathway in lipopolysaccharide-stimulated macrophage

Quercetin is a flavonoid molecule ubiquitous in nature and functions as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. Dose- and time-dependent effect of quercetin has been investigated on proinflammatory cytokine expression and NO production, focusing on its effects on the MAP kinases and the NF-B signal transduction pathways in LPS-stimulated RAW 264.7 cells by using RT-PCR and immunoblotting. Quercetin strongly reduced activation of phosphorylated ERK kinase and p38 MAP kinase but not JNK MAP kinase by LPS treatment. In addition, quercetin treatment inhibited NF-B activation through stabilization of the NF-B/IB complex and IB degradation and proinflammatory cytokines and NO/iNOS expression. Quercetin may exert its anti-inflammatory and immunomodulatory properties in the effect molecules such as proinflammatory cytokines and NO/iNOS by suppressing the activation of ERK and p38 MAP kinase, and NF-B/IB signal transduction pathways.     

Structure of the majorO-glycosidic oligosaccharide of monkey erythrocyte glycophorin

Sialic acids and the majorO-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey,Macaca fuscata) erythrocyte membranes were characterized.N-Glycolylneuraminic acid (neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. ThreeO-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose andN-acetylgalactosaminitol in the molar ratios of 111 (trisaccharide), 211 (tetrasaccharide) and 111 (pentasaccharide). The content of oligosaccharide units was estimated to be 1125 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gc2-3Gal1-3[Neu5Gc2-6]GalNAcol.     

Multiple shoot regeneration from root organ cultures of Populus alba x P. grandidentata

Excised roots of various ages from Crandon and Hansen clones of Populus alba x P. grandidentata were tested for their regeneration capacity. Sixty-day-old excised roots that contained root tips were found to be most suitable. The highest number of shoots (an average of 111 shoots/root segment with Crandon and 98 with Hansen) was obtained by adding 22M and 14M zeatin to the medium, respectively. The two clones of hybrid poplar responded similarly to growth regulator treatments; however, the number of shoots produced was greater from the root organs derived from Crandon clones. Regenerated shoots were rooted in basal Woody Plant Medium without any growth regulators. Successful transplantation into soil and growth was achieved with all plants.     

On age morphological changes of males of Chydoridae (Cladocera)

Summary Young and adult males of 11 species of Chydoridae are studied, their figures being published here (fig. 1–15). The necessity is stressed to distinguish young forms of males and gynandromorphic individuals.Pleuroxus balatonicus is considered to be described from the population ofPleuroxus unicatus having under Balaton Lake conditions retarded transformation of young males into adult form, and accordingly having unusually numerous young males. \qO\qs\qn\qo\qv\qn\qy\ye \qr\ye\qz\qu\ql\Qj\qt\qa\qt\qy 11 (. 1–15). . , Pleuroxus uncinatus , Pleuroxus balatonicus.     

Complete protection against melanoma in absence of autoimmune depigmentation after rejection of melanoma cells expressing α(1,3)galactosyl epitopes

The major barrier for xenotransplantation in humans is the presence of (1–3) Galactosyl epitopes (Gal) in xenogeneic tissue and the vast quantities of natural antibodies (Ab) produced by humans against this epitope. The binding of anti-Gal Ab to cells expressing Gal triggers a complement-mediated hyperacute rejection of target cells. The hyperacute rejection of whole cancer cells, modified to express Gal epitopes, could be exploited as a new cancer vaccine to treat human cancers. We tested this hypothesis in Galactosyltransferase knockout (GT KO) mice which, like humans, do not express Gal on their cell surfaces and can produce anti-Gal Ab. Forty-five percent of mice with preexisting anti-Gal Ab rejected Gal positive melanoma cells (B16Gal). These mice remained tumor-free for more than 90 days. The majority of control mice injected with B16Null, Gal negative cells succumbed to melanoma. The rejection of B16Gal induced strong long-lasting antitumor immunity against B16Null measured by the expansion of cytotoxic T lymphocytes. In addition, mice rejecting B16Gal were protected against melanoma since they survived a second rechallenge with B16Null. Protected mice developed antitumor immunity in the absence of autoimmune depigmentation (vitiligo). These results show that rejection of Gal positive melanoma cells can efficiently boost the immune response to other tumor associated antigens present in Gal negative melanoma cells. This study supports the concept of a novel anticancer vaccine to treat human malignancies.     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.