Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in Citrus leaf extracts

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) has been purified from orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] leaves using sucrose gradient centrifugation in a fixed angle rotor. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands corresponding to the two subunits of RuBP carboxylase were found. The large subunit coincided with the polypeptide band that has been previously reported to be preferentially mobilized during the spring and summer flush periods.
The degradation of RuBP carboxylase during autodigestion of Citrus leaf extracts, investigated by SDS-PAGE, occurred mainly at acidic (2.5-5.5) pH. The two subunits showed differences in the rate of degradation, the smaller being more rapidly hydrolyzed than the larger. At least four proteolytic activities were identified by means of inhibitor experiments: 1) a pepstatin A-sensitive activity that acts on both RuBP carboxylase subunits, 2) a mercurial ( p -hydroxymercuribenzoate and p -chloromercuriphenylsulfonate)-sensitive activity that degrades only the small subunit, 3) an EDTA-sensitive activity that hydrolyzes both the large and small subunits, and 4) a mercurial-stimulated activity that acts only on the large subunit. It is suggested that the last two proteases may be responsible for the degradation of RuBP carboxylase observed in vivo during the periods of mobilization of leaf protein in Citrus .     

Usefulness of manufactured tomato extracts in the diagnosis of tomato sensitization: Comparison with the prick-prick method

Background

Commercial available skin prick test with fruits can be negative in sensitized or allergic patients due to a reduction in biological activity during the manufacturing process. Prick-prick tests with fresh foods are often preferred, but they are a non-standardized procedure. The usefulness of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method in the diagnosis of tomato sensitization has been analyzed. The objective of the study was to assess the potential diagnostic of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method.

Methods

Two groups of patients were analyzed: Group I: 26 individuals reporting clinical symptoms induced by tomato contact or ingestion. Group II: 71 control individuals with no symptoms induced by tomato: 12 of them were previously skin prick test positive to a tomato extract, 39 were atopic and 20 were non-atopic. All individuals underwent prick-prick with fresh ripe peel Canary tomatoes and skin prick tested with freeze-dried peel and pulp extracts obtained from peel and pulp of Canary tomatoes at 10 mg/ml. Wheal sizes and prick test positivity (≥ 7 mm²) were compared between groups.

Results

In group I, 21 (81%) out of 26 patients were prick-prick positive. Twenty patients (77%) had positive skin prick test to peel extracts and 12 (46%) to pulp extracts. Prick-prick induced a mean wheal size of 43.81 ± 40.19 mm² compared with 44.25 ± 36.68 mm² induced by the peel extract (Not significant), and 17.79 ± 9.39 mm² induced by the pulp extract (p < 0.01). In group II, 13 (18%) out of 71 control patients were prick-prick positive. Twelve patients (all of them previously positive to peel extract) had positive skin prick test to peel and 3 to pulp. Prick-prick induced a mean wheal size of 28.88 ± 13.12 mm² compared with 33.17 ± 17.55 mm² induced by peel extract (Not significant), and 13.33 ± 4.80 mm² induced by pulp extract (p < 0.05 with peel extract and prick-prick).

Conclusion

Canary peel tomato extract seems to be as efficient as prick-prick tests with ripe tomatoes to diagnose patients sensitized to tomato. The wheal sizes induced by prick-prick and peel extracts were very similar and showed a high correlation coefficient.     

Cell separation from high cell density broths of Alcaligenes eutrophus by using a coagulant

Summary Alcaligenes eutrophus was successfully recovered from high cell density broths by pre-treatment with polyaluminium hydroxide chloride silicate as a coagulant at 36–90 mg Al/l. The optimum pH range for cell coagulation was 10–12. Subsequent centrifugation (45×g) and filtration (pore size 0.5 mm) gave a cell recovery of higher than 90%. The energy demand for cell recovery with the coagulant was only 3–11% of that without it.     

Proteolysis in vitro as the Way of Modification of the Protein Trophic Component

The protein hydrolysates of two types, fodder and for microbiological medium, have been obtained in vitro, using protein containing wastes of Iceland scallop fishery (WSF) and enzyme preparation from the red king crab hepatopancreas. The degree of protein degradation and composition of obtained hydrolysates were analyzed. Intensive protein hydrolysis was necessary to obtain the microbiological diagnostic cultural media that fit trophic requirements for 12 microbial test-cultures. Addition of the fodder protein hydrolysate to diet of juvenile salmons Salmo salar during mixed feeding produces a positive effect on their survival; the 5 and 20% replacement of fish flour by the fodder hydrolysate results in a reduction of fish mortality by 21 and 57%, respectively. The living weight of chickens increased by 15% after substitution of the 10% fish flour by the fodder WSF hydrolysate in their diet. The results obtained can be explained by a more effective assimilation of partly hydrolyzed proteins in comparison with native ones.     

Proteolysis in the quiescent seed

The importance of the proteolytic system of the quiescent seed in regard to mobilization of storage protein was assessed by identification of proteases already present at this stage. Extracts of quiescent cotyledons of white lupin ( Lupinus albus ) submitted to 45 h autolysis in vitro displayed a higher degree of protein degradation than that observed in vivo after two days of seed imbibition. Differences in the susceptibility to proteolysis were verified by densitometric analysis of the polypeptides after electrophoretic separation. The pH dependence of the proteolytic activities and the responses to specific protease inhibitors showed that the proteolytic systems vary from quiescent to 1 - to 3-day-imbibed cotyledons. By labelling an endogenous globulin with ¹²⁵I a sensitive radiometric assay allowed the identification of both an acidic and a neutral proteolytic system in the quiescent cotyledon. Within the quiescent seed there already exists a high potential for initiating proteolysis, so that the requirement for proteolysis by specific endopeptidases synthesized de novo upon imbibition only applies to part of the reserve proteins.     

Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry

Neurosecretory systems are known to synthesize and secrete a diverse class of peptide hormones which regulate many physiological processes. The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules--neuropeptides. As an ongoing effort to define the peptidome of the crustacean SG, we determine the neuropeptide complements of the SG of the Jonah crab, Cancer borealis, and the Maine lobster, Homarus americanus, using nanoflow liquid chromatography electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS. Numerous neuropeptides were identified, including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), beta-pigment dispersing hormone (beta-PDH), proctolin and HL/IGSL/IYRamide. Among them, two novel orcokinins were de novo sequenced from the SG of H. americanus. Three CPRPs including a novel isoform were sequenced in H. americanus. Four new CPRPs were sequenced from the SG of C. borealis. Our results show that structural polymorphisms in CPRPs (and thus the CHH precursors) are common in Dendrobranchiata as well as in Pleocyemata. The evolutionary relationship between the CPRPs is also discussed.     

Proteolysis: from the lysosome to ubiquitin and the proteasome

How the genetic code is translated into proteins was a key focus of biological research before the 1980s, but how these proteins are degraded remained a neglected area. With the discovery of the lysosome, it was suggested that cellular proteins are degraded in this organelle. However, several independent lines of experimental evidence strongly indicated that non-lysosomal pathways have an important role in intracellular proteolysis, although their identity and mechanisms of action remained obscure. The discovery of the ubiquitin-proteasome system resolved this enigma.     

Polymorphism in high-affinity calcium-binding proteins from crustacean sarcoplasm

The sarcoplasmic calcium-binding proteins (SCP) from crayfish, lobster and shrimp myogen have been purified to homogeneity. These proteins exist as dimers and dissociate in the presence of sodium dodecyl sulfate or urea in subunits of 22000 molecular weight. During the last step of purification (DEAE-cellulose chromatography), SCP emerges in three peaks in the ratio of 14:1.5:1 for crayfish, of 7:2:1 for lobster and of 3:2:1 for shrimp. Gel electrophoresis and isoelectrofocusing experiments, under native and denaturing conditions, indicate that among the three SCP isotypes there are only two different polypeptide chains, alpha and beta, which appear in the form of three dimers: alpha 2, alpha beta and beta 2. The alpha and beta subunits differ slightly in polypeptide chain composition as found by amino acid analyses of the crayfish and lobster SCPs, and also by comparison of tryptic peptides for crayfish SCPs. The polymorphism observed in crustacean SCPs, which is increased by their ability to form dimers, contrasts with the situation prevailing among other invertebrate SCPs and vertebrate parvalbumins where only monomeric isotypes are found. Equilibrium binding studies show that all three SCP isotypes from both crayfish and lobster display the same metal-binding properties. They have in their dimeric form six Ca2+-binding sites: two calcium-specific sites, two Ca/Mg sites that interact with positive cooperativity and two Ca/Mg sites that interact with negative cooperativity. Interactions between the two subunits of SCP seem to result in cooperative binding of Ca2+, which in turn may control more efficiently Ca2+ fluxes in crustacean muscle.     

Exocytosis of neurosecretory granules from the crustacean sinus gland in freeze-fracture

Exocytosis is clearly shown in freeze-fracture preparations to be the mechanism for neurosecretion granule release from axon endings in the crayfish sinus gland. The cytoplasmic leaflet (A-face) of axon ending membrane is characterized by randomly situated depressions representing invaginations of the axolemma, which are in contact with limiting membranes of neurohormone granules in the subjacent cytoplasm. The extracellular leaflet (B-face) of the axolemma at release sites exhibits complementary volcano-shaped protrusions which are cross-fractures through necks of channels formed by invaginating plasma membrane in contact with underlying neurosecretion granules. Structural variation in B-face protrusions is consistent with a spectrum of exocytotic profiles in various stages of formation, and with granules at different stages of passage out of the endings. Evidence in this study suggests that formation of exocytotic structures may begin by alteration of axon membrane structure at the neurosecretory ending-hemolymph interface prior to contact of the neurohormone granules with the axolemma. Limiting membranes of neurosecretory granules exhibit protrusions which appear to interconnect granules adjacent to release sites and to attach granules to the axolemma. Freeze-fracture is clearly shown to be an invaluable tool for monitoring the degree of exocytosis exhibited by sinus glands under normal conditions and under experimental acceleration of hormone release. This technique is capable therefore, of detecting slight increases in numbers of exocytotic profiles much more quickly and accurately than the examination of random thin sections.     

A miniature Clark-type oxygen electrode using a polyelectrolyte and its application as a glucose sensor

A miniature Clark-type oxygen electrode was fabricated by anisotropically etching silicon. A two-gold-electrode configuration was used and a double-layered gas-permeable membrane was formed directly on the electrolyte, poly(vinyl-4-ethylpyridinium bromide) in the sensitive area. These materials improved the electrode's stability in long-term storage and sterilization tolerance to a practical level. The 90% response time averaged 80 s and residual current 10%, with a good linear calibration curve. The oxygen electrode was also used to make an integrated sensor for the simultaneous determination of glucose and oxygen. The glucose sensor's response time was 50-110 s, with good linearity in glucose concentrations between 56 microM and 1.1 mM at 37 degrees C, pH 7.0.     

Chicory extracts from Cichorium intybus L. as potential antifungals

In this work extracts from roots of the common vegetable Cichorium intybus L., highly appreciated for its bitter taste, were studied to investigate their possible biological activity on fungi from a variety of ecological environments: some are parasites on plants (phytopathogens) or of animals and humans (zoophilic and anthropophilic dermatophytes), others live on the soil and only seldom parasitize animals (geophilic dermatophytes). The extracts were ineffective on geophilic species and on tested phytopathogens, with the exception of Pythium ultimum, whereas they inhibited the growth of zoophilic and anthropophilic dermatophytes, in particular Trichophyton tonsurans var. sulfureum, whose treatment caused morphological anomalies, here observed by scanning electron microscopy. This behaviour is discussed on the basis of the presence in the chicory extract of the two main sesquiterpene lactones, 8-deoxylactucin and 11β,13-dihydrolactucin.