Protease activity inAgaricus bisporus during periodic fruiting (flushing) and sporophore development

Protease activity from sporophores and mycelium of the mushroomAgaricus bisporus was assayed during periodic cropping (flushing) and from sporophores during maturation. When the sporophores were harvested at the same developmental stages (pins or buttons) during cropping, proteolytic activity of the sporophores was found to oscillate with the same periodicity as the flushing cycle. For pin mushrooms (an early stage of development), peaks of activity occurred during the interflush periods, whereas for button mushrooms (a later stage of development) peak proteolytic activity coincided with the periods of maximum production. The proteolytic activity in the mycelium remained low and varied little with time. Of the tissues within the sporophore, gill tissue had a higher activity than the stipe or pileus. The changes in activity during sporophore development or maturation depended on the period in the flushing cycle when the sporophore was initiated. The results are discussed in relation to the possible role and regulation of flush co-ordinated proteases.     

FluG and flbA function interdependently to initiate conidiophore development in Aspergillus nidulans through brlA beta activation.

The Aspergillus nidulans fluG gene is necessary for the synthesis of a small diffusible factor that is required for the endogenously regulated induction of asexual sporulation that takes place during the development of an air-exposed colony. Previous work established that FluG is present at nearly constant levels throughout the Aspergillus life cycle, leading to the hypothesis that FluG factor is constitutively produced and development initiates after its concentration surpasses a fixed threshold. Here we show that overexpression of fluG can overcome the developmental block normally imposed on vegetative cells in submerged culture and leads to the formation of complex conidiophores that are remarkably similar to wild-tye conidiophores made by air- exposed colonies. This fluG-induced sporulation requires the activities of other early developmental regulatory genes including, flA, flB, flC, flD, flE, and brlA. The requirement for flbA in fluG-induced sporulation is particularly interesting because overexpression of flbA can also induce sporulation in submerged culture and this flbA activity requires fluG. The interdependence of fluG and flbA activities suggests a close relationship between the products of these two genes in controlling conidiophore development. In addition to the endogenous sporulation signal provided by fluG, several environmental factors, including air exposure, carbon or nitrogen stress, and increased osmolarity, can influence developmental activation. We demonstrate that each of these signals requires the brlA beta gene, but not brlA alpha, to initiate conidiophore development. We present a model to account for the complex genetic and environmental controls leading to the activation of brlA beta and sporulation.     

The production of toxic protease by the entomopathogenous fungus Metarhizium anisopliae in submerged culture

The production of a toxic complex of proteolytic enzymes by Metarhizium anisopliae was evaluated with 29 nitrogen sources in modified Czapek-Dox medium in submerged cultures. The proteolytic complex is more constitutive than that of Beauveria bassiana and its production is influenced by the quality of complex natural media. The highest activity was attained with Galleria mellonella proteins. The proteolytic complex manifests proteolytic activity of two pH optima, 5.5 and 8.0. The ratio of these two activities differs markedly with the nitrogen source used, but the major proteolytic activity occurs at pH 5.5.     

Protease-producing psychrotrophic bacteria isolated from Antarctica

The extracellular protease production capacity of 840 bacterial strains isolated during the austral summers of 1989/90 and 1991/92 from different sources of the Antarctic ecosystem was analysed in skim-milk agar plates. Thirty-four psychrotrophic strains were selected, classified at genus level and tested from proteolytic activity by the azocasein method from the cell-free supernatant of submerged cultures. Thirty-two of the selected strains were Gram-negative bacteria and Pseudomonas was the predominant genus. Three Pseudomonas maltophilia strains showed the highest levels of proteolytic activity at 20°C. No correlation was observed between the proteolytic activity estimated by the ring of hydrolysis in skim-milk agar plates and the activity measured by the azocasein method. The results suggest that these psychrotrophic strains are potentially useful for developing a biotechnological process to produce proteases with high activity at moderate temperatures.     

Role of autocide AMI in development of Myxococcus xanthus.

A new developmental mutant of Myxococcus xanthus has been isolated by screening TnV insertion mutants for AMI-dependent development in submerged culture. This mutant (ER304) aggregated and sporulated on agar surfaces but required at least 3.8 micrograms of autocide AMI per ml for development in submerged cultures. Spore rescue of ER304 was obtained with the saturated, monounsaturated, and diunsaturated fatty acid fractions of AMI, with specific activities of 68, 115, and 700 U/mg, respectively. In addition, several model fatty acids were capable of rescuing sporulation of ER304; however, there was no correlation between specific lytic activity observed in vegetative cultures and specific rescue activity. Rescue of ER304 was effected during the first ca. 12 h after the initiation of starvation conditions; after this time, addition of AMI or model fatty acids killed the cells. Supernatant fluids of ER304 rescued development in dsg mutants (e.g., DK3260) in submerged cultures, but dsg mutant supernatant fluids were incapable of rescuing ER304 development. The data presented in this article support the idea that the primary mechanism of rescue by AMI is not via lysis, although developmental lysis may be an indirect result of the rescue event. A membrane permeability model is presented to explain the role of autocides in early developmental events in wild-type strains and in the aggregation and sporulation rescue of developmental mutants ER304 and DK3260.     

Production of Proteases from Brevibacterium Linens

Protease formation in submerged cultivations of Brevibacterium linens was studied. The effect of several proteinaceous materials on the production of proteolytic enzymes was investigated in mineral media containing 0.2% malt extract for bacterial growth. The addition (0.5%) of yeast extract or enzymatically hydrolyzed casein considerably increased the amount of protease formed, whereas ammonium salts supplied additionally in most cases had a repressive effect on enzyme formation. Furthermore, the kinetic of protease formation was determined in a highly instrumented fermenter system. Respiration activity indicated several phases of bacterial growth. Most of the proteolytic activity was synthesized during active growth; there was only a small increase in the stationary phase. A total proteolytic activity of 36 U/ml was formed in 24 hr. Concentration of α-amino nitrogen decreased steadily and ammonium ions accumulated during bacterial growth. Electrophoretic analysis revealed the occurrence of one leucine aminopeptidase (26 kDa monomer) and several proteases. There is a broad spectrum of proteolytic active proteins in the range of 11-66 kD which may be caused by some auto-degrading effects.     

Cotranslational Proteolysis Dominates Glutathione Homeostasis to Support Proper Growth and Development

The earliest proteolytic event affecting most proteins is the excision of the initiating Met (NME). This is an essential and ubiquitous cotranslational process tightly regulated in all eukaryotes. Currently, the effects of NME on unknown complex cellular networks and the ways in which its inhibition leads to developmental defects and cell growth arrest remain poorly understood. Here, we provide insight into the earliest molecular mechanisms associated with the inhibition of the NME process in Arabidopsis thaliana. We demonstrate that the developmental defects induced by NME inhibition are caused by an increase in cellular proteolytic activity, primarily induced by an increase in the number of proteins targeted for rapid degradation. This deregulation drives, through the increase of the free amino acids pool, a perturbation of the glutathione homeostasis, which corresponds to the earliest limiting, reversible step promoting the phenotype. We demonstrate that these effects are universally conserved and that the reestablishment of the appropriate glutathione status restores growth and proper development in various organisms. Finally, we describe a novel integrated model in which NME, protein N-α-acylation, proteolysis, and glutathione homeostasis operate in a sequentially regulated mechanism that directs both growth and development.     

The mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions.

In the studies reported here, we have examined the properties of the Mcp element from the Drosophila melanogaster bithorax complex (BX-C). We have found that sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and that they silence adjacent reporters by a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a "pairing-sensitive" silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals

Although maize endosperm undergoes programmed cell death during its development, it is not known whether this developmental feature is common to cereals or whether it arose inadvertently from the selection process that resulted in the enlarged endosperm of modern maize. Examination of wheat endosperm during its development revealed that this tissue undergoes a programmed cell death that shares features with the maize program but differs in some aspects of its execution. Cell death initiated and progressed stochastically in wheat endosperm in contrast to maize where cell death initiates within the upper central endosperm and expands outward. After a peak of ethylene production during early development, wheat endosperm DNA underwent internucleosomal fragmentation that was detectable from mid to late development. The developmental onset and progression of DNA degradation was regulated by the level of ethylene production and perception. These observations suggest that programmed cell death of the endosperm and regulation of this program by ethylene is not unique to maize but that differences in the execution of the program appear to exist among cereals.     

Mcp4, a meiotic coiled-coil protein, plays a role in F-actin positioning during Schizosaccharomyces pombe meiosis

Some meiosis-specific proteins of Schizosaccharomyces pombe harbor coiled-coil motifs and play essential roles in meiotic progression. Here we describe Mcp4, a novel meiosis-specific protein whose expression is abruptly induced at the horsetail phase and which remains expressed until sporulation is finished. Fluorescence microscopic analysis revealed that Mcp4 alters its subcellular localization during meiosis in a manner that partially resembles the movement of F-actin during meiosis. Mcp4 and F-actin never colocalize; rather, they are located in a side-by-side manner. When forespore membrane formation begins at metaphase II, the Mcp4 signals assemble at the lagging face of the dividing nuclei. At this stage, they are sandwiched between F-actin and the nucleus. Mcp4, in turn, appears to sandwich F-actin with Meu14. In mcp4Delta cells at anaphase II, the F-actin, which is normally dumbbell-shaped, adopts an abnormal balloon shape. Spores of mcp4Delta cells were sensitive to NaCl, although their shape and viability were normal. Taken together, we conclude that Mcp4 plays a role in the accurate positioning of F-actin during S. pombe meiosis.     

Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.     

The chemorepulsive activity of secreted semaphorins is regulated by furin-dependent proteolytic processing.

The semaphorins are a large group of cell surface and secreted proteins implicated in axonal pathfinding. Here we show that the secreted mouse semaphorin D (SemD) is synthesized as an inactive precursor (proSemD) and becomes repulsive for sensory and sympathetic neurites upon proteolytic cleavage. ProSemD processing can be blocked completely by an inhibitor selective for furin-like endoproteases or mutagenesis of three conserved dibasic cleavage sites. Its C-terminal pro-peptide contains a processing signal that is essential for SemD to acquire its full repulsive activity. SemD processing is regulated during the embryonic development of the mouse and determines the magnitude of its repulsive activity. Similarly to SemD, the secreted semaphorins SemA and SemE display repulsive properties that are regulated by processing. Our data suggest that differential proteolytic processing determines the repulsive potency of secreted semaphorins and implicate proteolysis as an important regulatory mechanism in axonal pathfinding.     

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.     

Functional interaction between an insulator and a Pc-dependent silencer by the example of the Mcp boundary of the <Emphasis Type="Italic">Drosophila melanogaster Abd-B</Emphasis> gene

Insulators are cis-regulatory elements that prevent improper gene activation and heterochromatin spreading. As shown previously, the boundary element, Mcp, from the regulatory region of the Drosophila melanogaster Abd-B gene, contains insulator. Here, we studied the boundary function of the Mcp insulator and showed that this function is provided by two modules. One is responsible for long-distance interactions and the capability of blocking enhancers. The other is essential for blocking Pc-dependent repression. It was observed for the first time that an insulator increased the repressor activity of a neighbor silencer.     

Co-ordinate regulation of sterol biosynthesis enzyme activity during accumulation of sterols in developing rape and tobacco seed

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol methyl transferase 1 and sterol acyltransferase, key enzymes involved in phytosterol biosynthesis were shown to be co-ordinately regulated during oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.) seed development. In both plants, enzyme activities were low during the initial stages of seed development, increasing towards mid-maturation where they remained stable for a time, before declining rapidly as the oilseeds reached maturity. During seed development, the level of total sterols increased 12-fold in tobacco and 9-fold in rape, primarily due to an increase in steryl ester production. In both seed tissues, stages of maximum enzyme activity coincided with periods of high rates of sterol production, indicating developmental regulation of the enzymes to be responsible for the increases in the sterol content observed during seed development. Consistent with previous studies the data presented suggest that sterol biosynthesis is regulated by two key steps, although there may be others. The first is the regulation of carbon flux into the isoprenoid pathway to cycloartenol. The second is the flux from cycloartenol to Delta(5)-end-product sterols. The implications of the results in terms of enhancing seed sterol levels by genetic modification are also discussed.