Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

Rats that consume caffeine show decreased brain protein synthesis

The effect of caffeine on protein synthesis in brain and liver was studied. When caffeine was added to a post-mitochondrial supernatant from rat brain protein synthesis was inhibited, i.e. 1 mM caffeine about 20%. The effect on protein synthesis of two weeks administration of large doses of caffeine in the drinking fluid of rats was also measured. Caffeine decreased protein synthesis in rat brain by about 32% and 20% compared with ad libitum and pair-fed controls. Protein synthesis was calculated taking into account the levels of free leucine determined by HPLC: 0.10 mol/g brain of ad libitum; 0.11 for pair-fed and 0.07 for caffeine. The pattern of proteins synthesized was not significantly altered by caffeine as shown by gel-electrophoresis and fluorography. There was no effect on protein synthesis of liver. The possible significance of these results is briefly discussed.     

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking

We have previously shown that mitochondrial membrane potential () drop promoted by prooxidants and Ca²⁺ can be reversed but not sustained by ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after was collapsed. Total recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during drop continues even after is already eliminated. Titration with 5,5-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

The NGF complex from the African ratMastomys natalensis

The 7S NGF complex from the male mouse submaxillary gland consists of the , and subunits in the ratio ₂₂. The (NGF) subunit contains all the known biolocial activity of 7S NGF. The and subunits are both members of glandular kallikrein gene family, yet only subunit has protease activity. The subunit plays a role in the processing of preproNGF to its mature form, while the role of the subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the or subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the ratMastomys natalensis in order to more fully understand the roles of the and subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio ₂. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.Special issue dedicated to Dr. Lawrence Austin     

Acute diabetes modulates response to ischemia in isolated rat heart

Role of protein kinase C (PKC) isotypes in the regulation of neutrophil function are not clearly known. In the present study we purified the -PKC and -PKC isotypes from human neutrophil. Both the isotypes are immunoreactive only to their respective antibodies. -PKC was further confirmed by RT-PCR using specific primer. Co-factor requirements for both the kinases were found to be different when DG and ceramide were used as second messenger. Selective substrate specificities were determined for both and -PKC using isotype specific pseudosubstrates viz., [Ser²⁵]PKC [19-31] and [Ser¹¹⁹]PKC[113-130] respectively. Endogenous protein phosphorylation by purified -PKC and -PKC showed their functional differences in neutrophil. -PKC phosphorylated 13, 15, 19, 33, 36, 47, 80 and 92 kDa proteins and -PKC phosphorylated 19, 22, 42, 47, 75 and 87 kDa proteins, only exception was the phosphorylation of 47 kDa protein which had been phosphorylated by both the kinases. Differences in phosphorylation between -PKC and -PKC clearly indicate the selective role for these PKC isotypes in the activation sequences of neutrophil.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Expression of storage-protein genes during soybean seed development

Mature seeds of Glycine max (L.) Merr. contain two major storage proteins, a glycosylated 7S protein (conglycinin) and a non-glycosylated 11S protein (glycinin). Accumulation of these proteins and their mRNAs during seed development in cv. Provar was studied by SDS polyacrylamide gel electrophoresis and by Northern (DNA-RNA) hybridization. The 11S acidic and basic subunits and the 7S and subunits began to accumulate 18–20 d after pollination, shortly after the termination of cell division in developing cotyledons, whereas the 7S and 11S A-4 subunits were not detected until one to two weeks later, during the maturation phase of development. Messenger RNAs for 7S and 11S proteins were first detected 14–18 d after pollination, several days before the accumulation of storage proteins. Extracts from embryonic axes contained reduced levels of the 7S subunit, very little 11S protein, no detectable 7S or 11S A-4 subunits, and an additional 7S subunit not found in cotyledons. Soybean axes and cotyledons therefore differ in their synthesis of seed storage proteins.Abbreviations cDNA complimentary DNA - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

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

Enzyme-catalyzed synthesis of alkyl β-D-glycosides with crude homogenate ofSulfolobus solfataricus

Summary Crude homogenate of thermophilic archaebacteriumSulfolobus solfataricus, possessing a -glycosidase, has been used to synthesize different alkyl -D-glycosides starting from phenyl -D-glucoside, phenyl -D-galactoside and lactose as carbohydrate donors. High product yield (95% with respect to the carbohydrate donor) of octyl -D-glucoside has been obtained in a two-phase system containing 5% of water. The enantioselection for the galactosyl transfer to the secondary hydroxyl group of propane-1,2-diol is higher than that found using -galactosidase fromE. coli.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Anomeric preference of glucose utilization in rat erythrocytes

The -anomer of glucose relative to the -anomer was more rapidly metabolized into lactate by rat erythrocytes at 37° C (/ ratio = ca. 1.3): the amounts of - and -D-glucose metabolized into lactate during 3 min were 0.21 and 0.27 mol/gHb, respectively. Also, the transport of -D-glucose into erythrocytes was more rapid than that of -D-glucose: the amounts of - and -D-glucose transported into erythrocytes during 3 min were approximately 3.5 and 5.0 mol/gHb, respectively. Glucose phosphorylation by rat erythrocyte hexokinase (i.e., a possible rate-limiting step in glycolysis) occurred at higher velocities with the -anomer than with the a-anomer (/ ratio = 1.28). The Km value of hexokinase for either anomer of glucose was 53 M. The glucose concentrations in erythrocytes incubated with - and -D-glucose reached about 1 mM in 1 min, indicating that hexokinase is almost completely saturated with glucose within less than 1 min. The results suggest that glucose phosphorylation and glucose transport are major and minor determinants, respectively, for the anomeric preference of glucose utilization in rat erythrocytes.     

Triggering and predisposing factors in the “Red” decline syndrome of Norway spruce (Picea abies)

Summary Analysis of 62 mature Norway spruce (Picea abies provenance Viborg) trees growing in a Danish plantation was undertaken along with analysis of their nutrient contents (N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B and Na), in each of the three youngest needle age classes, from branches of four exposure directions near the tree top. The aim was to investigate if one among the studied possible predisposing factors was also a triggering factor in the 1989 outbreak of the Red Norway spruce decline in Denmark. Neither nutrient imbalance or deficiency, nor excessive N-deposition or salt-stress were indicated as triggering factors in 1989. The Red syndrome, noticeable for the bright red colour of the current-year needles, was found to be an extension of the European type Novel Decline. Red syndrome is similar to previously reported phenomena of top-dying and sub top-dying, in that it had fewer needle age classes and significantly higher contents of mobile cations (and Ca) in the younger needle classes. Tree ring analysis suggested that the Red syndrome was initiated in the early 1980s, when the trees experienced adverse climatic conditions. Because of this long-term development of the Red Norway spruce decline syndrome, it is concluded that a triggering factor is of minor importance relative to the multitude of predisposing factors.     

Synthesis of aspartame precursor by solid thermolysin in organic solvent

Summary The enzymatic synthesis of a peptide compound was carried out successfully in homogeneous organic solvent.Solid Thermolysin was found to catalyze the synthetic reaction of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z-APM; a precursor of sweetner Aspartame) from N-benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl ester (L-PheOMe) in a 98 percent organic medium (ethylacetatebenzenemethanolwater=5029192). The dissolution of enzyme was not observed. The optimal pH shifted to acidic side by 1.0 pH unit, compared with that in aqueous medium. The enzymatic activity of solid thermolysin with an average size of 3.4×9.5 m was determined to be 0.18 moles-product/(mg-solid)·h under the initial concentrations of L-PheOMe of 0.1M and Z-L-Asp of 0.05M, and at pH 6.0 and 40°C.     

The living factory: In vivo Production of N-acetyllactosamine containing carbohydrates in E. coli

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a (1,4)GlcNAc-transferase), and the Neisseria (1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as Gal(1,4)[GlcNAc(1,4)]₄GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for Gal(1,4)GlcNAc (1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.     

Mediterranean types of β-thalassemia in the German population

Summary Forty -thalassemia genes from unrelated German heterozygotes with no known foreign ancestry were examined using the oligonucleotide technique and DNA restriction analysis, with the aim of determining the contribution of Mediterranean -thalassemia mutations to the prevalence of this trait in the German population. Of the 40 -thalassemia genes, 26 were identified as Mediterranean types (20 39 nonsense, 3 IVS2 nt 110, 2 IVS2 nt1, 1 IVS1 ntl GA). The geographic distribution of the birthplaces of the probands' grandparents revealed no difference in the proportion of Mediterranean and unidentified -thalassemia genes in the west and the north of Germany.