Structural Changes of β-Lactoglobulin during Thermal Unfolding and Refolding – An FT-IR and Circular Dichroism Study

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of -lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly -sheet, still remained intact even at 90°C. Noticeable changes in secondary structure of -lactoglobulin were observed only above 70°C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded -lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded -lactoglobulin. The combined CD and FT-IR data indicated that refolded form of -lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.     

The use of norbornene derivatives in the synthesis of conformationally constrained peptides and pseudo-peptides

The desymmetrisation of endo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrisation adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating an endo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of -turns and parallel -sheets.     

Sequential changes in MHC antigen expression induced by the v-Ki-ras oncogene

A series of early-passage cell lines were transformed with the v-Ki-ras oncogene with the aim of examining the effect of an activatedras gene on the ability of these cells to express major histocompatibility complex (MHC) antigens. These cell lines were found to undergo multiple phenotypic changes upon transformation and subsequent proliferation. At early passage, the predominant effect ofras was an increased ability to express class II antigens when induced with interferon (IFN). For class I antigens, maximum levels of expression induced with IFN were largely unaffected, however, decreased sensitivity to induction with this lymphokine was noted. With subsequent in vitro or in vivo passage, both class I and class II antigen inducibility was attenuated. The latter phenotypic change was found to be transferable by coculture, implicating a soluble IFN antagonist. Conditioned media fromras-transformed cells treated to activate their latent transforming growth factor (TGF) content mediated similar changes in MHC antigen inducibility, suggesting that TGF\ may be involved in modulating MHC antigen expression inras-transformed cells.     

β-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether and derivatives by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 4-ethoxy-3-methoxyphenyl-glycerol--guaiacyl ether (V) in low nitrogen, stationary cultures under which conditions the ligninolytic enzyme system is expressed. 4-Ethoxy-3-methoxyphenylglycerol XIII, guaicol and 4-ethoxy-3-methoxybenzyl alcohol (II) were isolated as metabolic products. Exogenously added XIII was rapidly converted to 4-ethoxy-3-methoxybenzyl alcohol indicating that it is an intermediate in the metabolism of V. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-hydroxypropane VI. The degradation pathway for this dimer also included initial -ether cleavage and -hydroxylation of the diol product 1-(4-ethoxy-3-methoxyphenyl) 2,3 dihydroxypropane (XI) to yield the triol XIII which was cleaved at the , bond to yield 4-ethoxy-3-methoxybenzyl alcohol. Finally P. chrysosporium also cleaved the dimer 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-hydroxypropane (VIII) at the -ether linkage yielding 1-(4-ethoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IX) which was subsequently cleaved at the , bond to yield II. All of the results indicate that oxidative -ether cleavage is an important initial reaction in the metabolism of -aryl ether lignin substructure dimeric compounds. Metabolities were identified after comparison with chemically synthesized standards by gas liquid chromatography-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography     

Circadian changes in synaptic ribbons and spherules in pinealocytes of the Syrian hamster (Mesocricetus auratus)

Summary In the present study, synaptic ribbons were studied morphologically and quantitatively in hamster pineal gland. The number of ribbons and spherules of hamster pinealocytes was counted over a 24-h period. The 24-h variations in the quantity of synaptic ribbons were found to parallel fluctuations in pineal melatonin concentrations. No significant circadian changes were observed for synaptic spherules, indicating different roles for these two structures.     

Aluminum borate whisker-mediated production of transgenic tobacco plants

An aluminum borate whiskers-mediated transformation system for calluses of tobacco (Nicotiana tabacum, cv. SR-1) has been developed. A total of 50 small pieces of calluses were vigorously agitated in a liquid medium containing aluminum borate whiskers, pBI221 plasmid carrying the -glucuronidase (GUS) gene, and pBI222 plasmid carrying the hygromycin phosphotransferase (HPT) gene. After treatment, calluses were cultured to select for hygromycin resistance, and three resistant calluses were obtained. Adventitious shoots were produced from each hygromycin-resistant callus and were transferred to rooting medium. A total of three plantlets obtained from each hygromycin-resistant callus were acclimatized and established in soil. Polymerase chain reaction analysis revealed that all the plantlets were cotransformed with both the GUS and HPT genes. Detached leaves of transgenic individuals showed clear hygromycin resistance when cultured in liquid medium. Histochemical assay for GUS revealed that one of these transgenic plants expressed the GUS gene, indicating coexpression of foreign genes.     

Biosynthesis and Molecular Genetics of Clavulanic Acid

The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated. Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes. Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes. A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved. While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic -lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams. Conversion of clavaminic acid to clavulanic acid requires side chain modfication as well as inversion of ring stereochemistry. This stereochemical change occurs coincident with acquisition of the -lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance. In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack -lactamase inhibitory activity.     

Reproduction of,and ultrastructural changes induced by,Spiroplasma citri in sieve tube of citrus leaves

Summary Electron microscope examination of ultrathin sections of leaf veins of stubborn—affected citrus seedlings revealed three morphotypes ofSpiroplasma citri free in the cytoplasm of mature sieve elements. In addition to these, inclusions believed to beSpiroplasma citri, some in various stages of degeneration, were occasionally found inside spherical, ovoid, or angular membraneous structures (= packets) which occurred in sieve elements devoid of any recognizable organelles. These packets which varied in size from 1.0 to 1.8 m wide an 1.9 to 3.5 m long were bounded by unit membrane ca. 9 to 10 nm thick. Spiroplasmas and packets were apparently absent in sieve elements of leaf veins of healthy citrus seedlings. Three types of packets were recognized based on the size of spiroplasmas contained: type I packets contained large, intermediate, and small spiroplasmas, but small forms predominated; type II packets contained a mixture of large and intermediate forms, while type III packets contained essentially tightly—packed large forms. Results of the study suggested that the spiroplasma-containing packets are either definite reproductive structures peculiar toSpiroplasma citri or are sieve-tube cells in various stages of plasmolysis. Evidence is presented indicating that within a given packet small spiroplasmas were produced from large spiroplasmas by some process of cell constriction followed by fission, or by budding. Since these spiroplasma—containing packets were infrequently observed in infected tissues we suggest that cell division by budding, of by constriction followed by fission into unequal daughter cells may be the principal mode of reproduction inSpiroplasma citri.     

β-1-3- and β-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The -glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various -glucanases and by chromatography show that both -1-3- and -1-4-linkages are formed at high substrate concentrations. In the absence of MgCl₂, -1-3-linked glucans are mainly produced. By increasing MgCl₂ concentrations the total synthesis activity and -1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl₂, -1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl₂.Abbreviations G.S. I glucan synthetase I - G.S. II glucan synthetase II - Dol. P dolichol phosphate     

Receptor Interactions of β-N-Oxalyl-L-α,β-Diaminopropionic Acid,the Lathyrus sativus Putative Excitotoxin,with Synaptic Membranes

Direct evidence for the excitotoxicity of -N-oxalyl-L-,-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 ³H]ODAP ([³H]ODAP) to synaptic membranes. [³H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15–20% of the total binding) and was also not saturable. The low specific binding of [³H]ODAP remained unaltered under a variety of assay conditions. A low B_max of 3.2 ± 0.4 pmol/mg and K_d 0.2 ± 0.08 M could be discerned for the high affinity interactions under conditions wherein more than 80–90% of the binding was nonspecific. While ODAP could inhibit the binding of [³H]glutamate to chick synaptic membranes with a K_i of 10 ± 0.9 M, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [³H]glutamate. The very low specific binding of [³H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.     

Anti-(transforming growth factor β) antibodies with predefined specificity inhibit metastasis of highly tumorigenic human xenotransplants in nu/nu mice

Monoclonal antibodies (mAb) were prepared against conjugated transforming growth factor 1 (TGF1) peptides: amino acid positions 48–60 and positions 86–101. Two antibodies, mAb 16-3G1 [anti-(48–60)] and mAb 5-2G6 [anti-(86–101)] cross-reacted with native TGF1,-2 and-3 (16-3G1) or only with native TGF1 (5-2G6). Both mAb were used to characterize TGF-mediated effects on the metastatic potential in nude mice of human carcinoma cell line SLU-1 and its metastatic subline SLU-M1. Autocrine TGF1-mediated up-regulation of cell proliferation and its suppression by anti-TGF antibodies in vitro was recorded for SLU-M1 cells whereas SLU-1 cell proliferation in vitro appeared to be refractory to anti-TGF antibodies and exogenous TGF-1. However, the potential of s.c. tumours to develop distant metastases in nude mice was about the same for both cell lines. Development of primary tumours and distant metastases could be suppressed by treatment of mice with anti-TGF antibodies. Thus we assume that the metastatic potential of tumour cells is independent of TGF-mediated growth-regulation effects in vitro. The anti-TGF-induced suppression of tumour progression and metastasis in nude mice might rather result from stimulation of the immune surveillance. TGF-mediated autocrine down-regulation of MHC-unrestricted cytotoxicity of activated human monocytes and CD56⁺ LAK cells and its reversion by anti-TGF antibodies could be readily demonstrated. In all our experimental series, the neutralizing potential of both anti-TGF antibodies, though directed against opposite sites of the TGF1 molecule, was very similar.     

Metabolism of the lignin model compounds veratrylglycerol-β-guaiacyl ether and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium metabolized the lignin model compounds veratylglycerol--guaiacyl ether I and 4-ethoxy-3-methoxy-phenylglycerol--guaiacyl ether V in stationary culture under an atmosphere of 100% oxygen and under nitrogen limiting conditions. 2-(o-methoxyphenoxy)-ethanol VII was identified as a product of the metabolism of both substrates. Veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol IV were identified as metabolites of I and V respectively. Metabolites were identified after comparison with chemically synthesized standards by mass spectrometry. These results indicate the existence of an enzyme system capable of directly cleaving the etherated dimers I and V at the , bond. The additional identification of 2-(o-methoxyphenoxy)-1,3 propanediol IX as a metabolic product indicates that cleavage of the alkyl-phenyl bond of these dimers or their metabolites also occurs.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC Thin layer chromatography     

Mechanism involved in the response of granulated vesicles in the mouse pinealocyte to acute cold exposure

Summary Rudimentary cilia have been observed in muscle cells lining the tube feet of Ophioderma brevispinum (Ophiuroidea) and in muscle cells of the body wall and parapodial glands of Owenia fusiformis (Polychaeta). A diplosomal basal body is associated with each cilium. Striated rootlets are absent. This is the first report on rudimentary cilia in muscle cells of an echinoderm and an annelid.Supported by NSF grant #GB-42211 to R.M. Rieger. Costs of some photographic supplies were defrayed by a grant from Sigma Xi to S.L. Gardiner     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Methionine induction of ACV synthetase inCephalosporium acremonium

Summary Methionine markedly stimulates the biosynthesis of penicillin N and cephalosporin C inCephalosporium acremonium. Examination of intra- and extracellular ACV tripeptide in non-producing mutant N-2 showed that growth in the presence of methionine increased ACV accumulation. Direct measurement of ACV synthetase activity in a cell-free system indicated that the methionine effect was mainly due to induction of this first enzyme of the -lactam biosynthetic pathway, resulting in a corresponding increase in -lactam production in both a low-producing strain and a high-producing mutant.     

Identity of esterase-22 and egasyn,the protein which complexes with microsomal β-glucuronidase

Recent experiments have demonstrated that egasyn not only sequesters -glucuronidase in microsomes by forming high molecular weight complexes with -glucuronidase, but also has carboxyl esterase activity. We have found several new phenotypes of egasyn-esterase after electrophoresis and isoelectric focusing of liver homogenates and purified egasyn of inbred and wild mouse strains. Several phenotypes corresponded in relative mobility and relative isoelectric point among inbred strains to that recently reported for esterase-22 by Eisenhardt and von Deimling [(1982). Comp. Biochem. Physiol. 73B:719]. This genetic evidence, plus a wide variety of comparative biochemical and physiological data, indicates that egasyn is identical to esterase-22. Both parental types of egasyn isozymes are expressed in heterozygous F₁ progeny, suggesting that alterations in the egasyn structural gene are responsible for the altered isoelectric points. Also, egasyn is a monomer since no new esterase bands appear in F₁ progeny. The variants in isoelectric point of egasyn map at or near the egasyn (Eg) gene within the esterases of cluster 1 near Es-9 on chromosome 8.This work was supported by Grant GM-33559 from the National Institutes of Health.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

The design,synthesis and study of siderophore-antibiotic conjugates Siderophore mediated drug transport

Summary The use of conjugates of microbial iron chelators (siderophores) and antibiotics for illicit transport of antibiotics into cells is a potentially powerful method for the rational design of therapeutic agents. The structural complexity of most natural siderophores has impeded progress in this area. Described here are the design, syntheses and preliminary biological studies of several siderophore--lactam antibiotic conjugates. Both hydroxamic-acid-based and catechol-based conjugates with and without amino acid spacers to carbacephalosporins were synthesized and demonstrated to be effective inhibitors ofEscherichia coli X580. Mutant selection was noted for each class of conjugates. Mutants selected from exposure of theE. coli to the hydroxamate conjugates were susceptible to the catechol conjugates and vice versa. Combinations of hydroxamate-and catechol-carbacephalosporin conjugates were most effective inhibitors ofE. coli X580.     

Involvement of β 1,4 galactosyltransferase 1 and Galβ 1→4GlcNAc groups in human hepatocarcinoma cell apoptosis

1,4 galactosyltransferase 1 ( 1,4GT1) synthesizes Gal 14GlcNAc groups in N-linked sugar chains of animal glycoproteins, which have been demonstrated to play an important role in many biological events, including sperm-egg interaction, cell migration and mammalian embryonic development. In this study, the mRNA level of 1,4GT1 was found to increase greatly during the 7721 hepatocarcinoma cells apoptosis induced by cycloheximide. Ricinus Communis Agglutinin-I staining indicated generous increase of Gal 14GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of 1,4GT1, which also led to the increased Gal 14GlcNAc groups on the transfected cell surface. All the observations suggested that 1,4GT1 and Gal 14GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.     

2-Phenylethylamine-induced changes in catecholamine receptor density: Implications for antidepressant drug action

It is now established that (1) concentrations of 2-phenylethylamine (PEA) are greatly increased in brain following administration of monoamine oxidase inhibitor (MAOI) antidepressants; (2) PEA is a metabolite of the MAOI antidepressant phenelzine; and (3) PEA may be a neuromodulator of catecholamine activity. On the basis of these observations, the effects of long term increases in brain PEA on catecholamine receptors have been assessed. Both PEA and antidepressants induced a reduction in the behavioural response to the ₂ adrenoceptor agonist salbutamol. Radioligand binding measurements revealed that 28 day administration of PEA in combination with the type B MAOI (–)-deprenyl results in a decrease in the density of ₁ adrenoceptors but not ₂ adrenoceptors in rat cerebral cortex and cerebellum. (–)-Deprenyl alone also induced a significant decrease in ₁-adrenoceptors but when PEA was added to this treatment there was a further decrease in ₂-adrenoceptor density. Only changes in ₁ adrenoceptor density were evident following 28 day administration of MAOI antidepressants. PEA also induced a decrease in the density of D1-like dopamine (DA) receptors in the rat striatum. MAOI antidepressants induced a decrease in the density of both D1-like and D2-like DA receptors. These data are discussed in terms of a possible role of PEA-catecholamine interactions in antidepressant drug action.