Elevated levels of plasma lipofuscins in patients with chronic renal failure

The fluorescence excitation and emission spectra observed in plasma from patients with chronic renal failure were reproduced by the generation of soluble lipofuscins in normal plasma samples by incubation with mixtures of L-dopa, dopamine, L-norepinephrine, L-epinephrine, 3-hydroxy-DL-kynurenine and 3-hydroxy-anthranilic acid for 24 h at 37 degrees C. Relative fluorescence intensity measurements consistently showed elevated plasma levels of the soluble lipofuscins in chronic renal failure: the means (n = 27) were 73.9 +/- 33.4 (SD) and 71.1 +/- 14.8 at emissions 413 nm and 445 nm respectively, in contrast to those of normal plasma samples (n = 11), 18.2 +/- 5.3 and 23.1 +/- 5.6. The maximum or shoulder at approximately 413 nm represents soluble lipofuscin that can be generated from 3-hydroxyanthranilic acid and the maximum or shoulder at approximately 445 nm represents soluble lipofuscins derived from the precursors listed above and probably from other related precursors. Gravimetric measurements also showed elevated levels of melanins in the plasma samples of patients with chronic renal failure: 2.72 +/- 0.38 mg/ml (n = 16), as compared to normal values: 1.70 +/- 0.10 mg/ml (n = 6). In individual patients haemodialysis reduced the fluorescence intensities to a range of 65-99% and the melanin levels to a range of 86-99% of the pre-dialysis values.     

Possible metabolic basis for the developmental anomaly observed in in vitro culture,called ‘vitreous plants’

Previous studies have shown that the development anomaly encountered in meristem culture, known as vitreous plants, is due to deficient lignin synthesis. This anomaly can be cured by addition of phloridzin to the culture medium. This study examines the activities of some enzymes involved in the synthesis of lignins and of flavonoids in normal and in vitreous plants of two apple cultivars. The results showed that all enzymes were consistently less active in the vitreous plants. This agrees with previous studies made on the hydroxycinnamate: CoA ligase activity in Prunus avium (L.) meristem-derived plants. The study on the substrate specificity of the enzyme demonstrates that while its activity is lower in the vitreous plant, its conformation is identical with that of the normal plant; the substrate that is specific to enzyme extracts of both sources is para-coumaric acid.     

Two calcium‐binding chaperones from the fat body of the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin‐type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N‐globular domain, a P‐arm domain, and a highly charged C‐terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause‐related expression analysis in the whole body revealed an upregulation of both genes by post‐diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause‐related expression pattern in the entire body for both genes. Fat body‐specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post‐diapause. This study suggests that calcium‐binding chaperones play similar and possibly gender‐specific roles during diapause.     

Analysis of an insect neuropeptide, Schistocerca gregaria ion transport peptide (ITP), expressed in insect cell systems

We have produced an active form of Schistocerca gregaria ion transport peptide (ITP) in an insect cell expression system. Transformed Drosophila Kc1 cells secreted a form of ITP into the cell culture medium that was proteolytically cleaved correctly at the amino (N)-terminus. Concentrated culture supernatant from transformed Kc1 and Hi5 cells had high biological activity when tested on isolated locust ilea. Conversely, ITP expressed by baculovirus-infected Sf9 cells was larger in size and had decreased specific activity compared to ITP produced by Kc1 cells due to incorrect cleavage of the peptide at the N-terminus in the baculovirus system. This demonstrates how processing of the secreted foreign protein (ITP) expressed under the late polyhedrin promoter is compromised in a baculovirus-infected cell. Transient transformation of Kc1 cells results in supernatants containing two forms of ITP; one form (A) co-elutes with synthetic ITP and the other form (B) has reduced electrophoretic mobility. In contrast, in stably transformed Kc1 cell supernatant, ITP is expressed in a single form, which has the same electrophoretic mobility and specific biological activity as form A produced by transiently transformed Kc1 cells. Arch.     

Interaction of Sclerotinia sclerotiorum with Brassica napus: cloning and characterization of endo- and exo-polygalacturonases expressed during saprophytic and parasitic modes

Five major and several minor PG isoenzymes were identified in a Sclerotinia sclerotiorum isolate from Brassica napus by isoelectric focusing and pectin gel overlays. Using a combination of degenerate PCR and expressed sequence tags (ESTs) four endo-polygalacturonase (PG) genes, designated as sspg1d, sspg3, sspg5, and sspg6, and two exo-PG genes, ssxpg1 and ssxpg2, were identified. SSPG1d is a member of the PG gene family previously described by Fraissinet-Tachet et al. [Curr. Genet. 29 (1995) 96]. The mature SSPG1d is a neutral PG, whereas fully processed SSPG3, SSPG5, and SSPG6 are acidic enzymes. Under saprophytic growth conditions, sspg1d, sspg3, sspg5, and ssxpg1 expression was induced by pectin and galacturonic acid and subject to catabolite repression by glucose. Conditions could not be identified under which sspg6 or ssxpg2 were expressed well. Transfer of mycelia from liquid media to solid substrates induced expression of sspg1d suggesting that it may also be regulated by thigmotrophic interactions. Under pathogenic conditions, sspg1d was highly expressed during infection. sspg3 was also expressed during infection, albeit at lower levels than sspg1d, whereas sspg5, sspg6, and ssxpg1 were expressed only weakly.     

The SBASE domain sequence library,release 10: domain architecture prediction

SBASE (http://www.icgeb.trieste.it/sbase) is an on-line collection of protein domain sequences and related computational tools designed to facilitate detection of domain homologies based on simple database search. The 10th 'jubilee release' of the SBASE library of protein domain sequences contains 1 052 904 protein sequence segments annotated by structure, function, ligand-binding or cellular topology, clustered into over 6000 domain groups. Domain identification and functional prediction are based on a comparison of BLAST search outputs with a knowledge base of biologically significant similarities extracted from known domain groups. The knowledge base is generated automatically for each domain group from the comparison of within-group ('self') and out-of-group ('non-self') similarities. This is a memory-based approach wherein group-specific similarity functions are automatically learned from the database.     

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

Aspirin--the first hundred years

'Take two aspirin and call me in the morning', may be an old cliché deriding the medical profession, but the story of the first 100 years of aspirin suggests that this drug may be of benefit for a far greater range of ailments than those who synthesised it 100 years ago could have imagined.