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.     

Restricted T cell receptor V-β and J-β usage in T cells from interleukin-2-cultured lymphocytes of ovarian and renal carcinomas

Tumour-infiltrating lymphocytes (TIL) are often observed in human tumours and their presence has been correlated with a better prognosis. It has been suggested that TIL are enriched for tumour-specific cytotoxic cells, and TIL activated and expanded in vitro by interleukin-2 (IL-2) are currently used in the therapy of human cancer. We have studied the T cell repertoire in IL-2-expanded TIL cells from patients with ovarian and renal carcinoma using T-cell-receptor-V--specific monoclonal antibodies and a polymerase-chain-reaction-based Southern blot technique for analysis of J- usage. In TIL lines derived from three of nine patients with ovarian carcinomas and from two of eight patients with renal carcinomas, selective usage of the V-6 or V-5 T-cell receptor gene products was found. The majority of the cells were CD4⁺, with up to 40% of the T cells utilizing the same V- gene. T-cell lines derived from peripheral blood lymphocytes from patients or healthy donors contained normal levels of V- subsets. Only moderate levels of V-6⁺ T cells were detected from freshly isolated TIL and the increase of this subpopulation appeared as a result of in vitro culture. The level of clonal restriction, as measured by the usage of J- gene segments within the V-5 or V-6 families, was analysed using a recently developed technique based on the polymerase chain reaction. Evidence for restricted J- usage was detected only in TIL expanded in vitro, while this was not the case in freshly isolated tumour-derived lymphocytes or T cell lines obtained from peripheral blood lymphocytes. The presence of a population with biased T cell receptor expression in cells derived from tumour tissue could be explained by their activation in vivo as a result of contact with tumour antigens and should be taken into consideration when discussing the therapeutic efficiency of IL-2-expanded TIL.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Participation of the cholinergic system in the excessive grooming behavior induced by neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI)

The neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI) injected into the ventral tegmental area (VTA) or intraventricularly (icv) induces excessive grooming behavior (EGB) and motor activity (MA). Here, we studied whether the cholinergic system is involved in the NEI-induced behavior. The present results demonstrate that atropine, a general muscarinic antagonist, injected icv previous to NEI, suppresses the behavior provoked by icv injections of the peptide, whereas the prior icv injection of dyhidro--erythroidine, a general nicotinic antagonist, did not affect the EGB and MA induced by the peptide. From the experimental evidence, it is suggested that NEI may act specifically on a cholinergic afferent to dopaminergic cells. Also, the results appear to indicate that a neural target, different from the dopamine system, may be activated by the peptide to elicit behavioral changes, such as EGB.     

Metabolization of cyanogenic glucosides inHevea brasiliensis

Over 90% of the cyanogenic precursors ofHevea seeds is stored in the endosperm tissue. During seedling development most of the cyanogenic material is consumed to form noncyanogenic compounds. No gaseous HCN is liberated in the course of this process. The -glucosidase, responsible for the cleavage of cyanogenic glucosides and the key enzyme for cyanogenesis is widely distributed over all tissues. The highest enzyme activity of the HCN-metabolizing -cyanoalaninesynthase is found in young seedling tissues. It is concluded, that the cyanogenic glucosides must be transported and metabolized in the young, growing tissues.Lecture held at the Tagung der Deutschen Botanischen Gesellschaft in Vienna, September 1984.     

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.     

The two β-oxidation sites in pea cotyledons

Two sites for -oxidation of fatty acids in pea (Pisum sativum L.) cotyledons exist. One site is the microbody, the other the mitochondrion. Mitochondrial -oxidation of fatty acids is carnitine-dependent. The fatty acid permeates the membrane as palmitoylcarnitine which is formed from cytosolic-side palmitoyl-CoA by a carnitine palmitoyltransferase located on the exterior face of the inner mitochondrial membrane as a peripheral protein. A single-gated pore integral membrane translocator is proposed to exchange the palmitoylcarnitine for carnitine or acetylcarnitine across the membrane. An internal (matrix side) carnitine palmitoyltransferase then reforms palmitoyl-CoA which enters -oxidation and subsequently the tricarboxylic-acid cycle.     

Estradiol Protects Against Oxygen and Glucose Deprivation in Rat Hippocampal Organotypic Cultures and Activates Akt and Inactivates GSK-3β

Here we investigated the neuroprotective effect of 17-estradiol in an in vitro model of ischemia. We used organotypic hippocampal slice cultures, acute or chronically treated with 17-estradiol (10 nM), and exposed to oxygen and glucose deprivation (OGD). Cellular death was quantified by measuring uptake of propidium iodide (PI), a marker of dead cells. In OGD exposed cultures, treated only with vehicle, about 70% of the CA1 area of hippocampus was labeled with PI, indicating a great percentage of cellular death. When cultures were treated with 17-estradiol (acute or chronically), this cellular death was reduced to 15%. This effect was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that both, chronic and acute, treatments with 17-estradiol induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of GSK-3. Our results show a clear neuroprotective effect of 17-estradiol and suggest that this effect could involve PI3-K pathway.     

Cloning and comparative sequence analysis of the gene coding for isopenicillin N synthase in Streptomyces

Summary The genes coding for isopenicillin N synthase (IPNS) in Streptomyces jumonjinensis and S. lipmanii were isolated from recombinant phage lambda libraries using the S. clavuligerus IPNS gene as a heterologous probe. The S. jumonjinensis IPNS gene has an open reading frame coding for 329 amino acids, identical in size to that of the previously cloned S. clavuligerus IPNS gene. A partial nucleotide sequence was also determined for the S. lipmanii IPNS gene. Comparison of the predicted amino acid sequences of all three streptomycete IPNS proteins shows that they exhibit more than 70% similarity, close to that found in comparisons among fungal IPNS proteins and significantly greater than that found, approximately 60%, between Streptomyces and fungal IPNS proteins. We conclude that procaryotic and eucaryotic IPNS genes are subgroups of a single family of microbial IPNS genes. Hybridization probes prepared from IPNS genes of the above streptomycete species were used to detect analogous genes in eight other strains that included both penicillin and cephalosporin producers and non-producers. Each producer strain responded with all three probes implying the presence of an IPNS gene. Surprisingly, several non-producer strains also responded with one or two of the probes. Our results suggest that IPNS-related genes may be more prevalent in Streptomyces than previously believed.     

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml^–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R _MTet chromatographic migration rate in relation to that of maltotetraose     

Distribution of Fucoidan Hydrolases and Some Glycosidases among Marine Invertebrates

Thirty-three species of marine invertebrates from the Sea of Japan were analyzed for contents of fucoidan hydrolases and some glycosidases. Fucoidan hydrolase activity was assessed by examining the effect of animal tissue extracts on fucoidans from the two brown seaweeds Laminaria cichorioides and Fucus evanescens, which have different structural characteristics. The activity of glycosidases (-glucosidase, -galactosidase, -fucosidase, and -mannosidase) was determined using p-nitrophenyl derivatives of sugars as substrates. It was found that glycosidases and fucoidan hydrolases of different specificities are fairly widely distributed among marine invertebrates. Mollusks and some species of echinoderms and arthropods showed the highest enzymatic activity. This research will enable us to choose organisms for the separation and study of fucoidan hydrolases and glycosidases, which may be useful in determining the structure of fucoidans.     

Characterization of the dopamine stimulated adenylate cyclase in the pedal ganglia ofMytilus edulis: Interactions with etorphine,β-endorphin,DALA, and methionine enkephalin

The dopamine-stimulated adenylate cyclase activity was studied both in vivo and in vitro in the central nervous system of the bivalve mollusc Mytilus edulis. Dopamine, epinine, and apormorphine stimulated the enzyme system. Fluphenazine, haloperidol, chlorpromaxine, and to a lesser extent BOL inhibited the dopamine-stimulated adenylate cyclase. Etorphine, -endorphine, DALA, and methionine enkephalin depressed cyclic AMP levels. This phenomena was naloxone reversible. In addition, the opioids inhibited the stimulation of adenylate cyclase by dopamine. This phenomena was also naloxone reversible. The study demonstrates an interaction among dopamine, the opioids, and cyclic AMP.This work was partially supported by Grant 1-T32GM07641-01 from the M.A.R.C. Program of N.I.G.M.S. and Grant 1S06RR08171-01 by the Division of Research Resources and the N.I.M.H.     

Locus-control-region-coupled betaS Antilles- and alpha2-hemoglobin genes select for high alpha2-hemoglobin expression in adult transgenic mice

Two transgenic lines of mice were produced which contained the ^S _Antilles- and ₂-hemoglobin genes trandemly coupled to the micro locus control region (LCR). The LCR^S _Antilles2-hemoglobin transgenic mice expressed high levels of ₂-hemoglobin while ^S _Antilles-hemoglobin expression was virtually undetectable. Abundant ₂-hemoglobin protein was observed in the blood of transgenic mice, while ^S _Antilles-hemoglobin chains could not be detected. Transgenic red blood cells had substantially decreased sensitivity to osmotic lysis. Attempts to produce homozygotes containing the transgene were unsuccessful. The phenotype of these mice closely resembles that of -thalassemic mice. The LCR^S _Antilles2 transgenic mice demonstrate that if the LCR is coupled to the ^S _Antilles- and ₂-hemoglobin genes in tandem, only the distal ₂-hemoglobin gene is selected for expression to significant levels in adult mice. These results support a reciprocally competitive model for LCR-hemoglobin developmental switching.     

Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use

An easily scaled-up technique has been designed to purify -mannanase from Bacillus licheniformis. Using flocculation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recovery of 47% and a specific activity of 4341 U mg^–1protein. The enzyme had maximum activity at 60 °C and pH 7.0. It was stable at 50 °C and pH 6.0 for 6 h, but lost all of its activity when held at 70 °C and pH 6.0 for 1 h.     

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.     

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     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.