Production of acid proteinases byHumicola lutea immobilized in polyacrylamide gel

Summary Fungal spores ofHumicola lutea 120–5 were entrapped in 5% polyacrylamide gel and were cultivated for 44–48 h to form a mycelial network inside the beads. A dense mycelial growth also occurred on the surface of the beads. It was possible to reuse the immobilized mycelium for production of acid proteinases in 12 different batches without loss of mechanical stability. The inoculum size should be controlled prior to its transfer into fresh production medium. Maximal enzyme production exceeding the level of free cell fermentation was registered in the fourth to seventh cycles. According to the size of the inoculum, half of the initial production rate was reached after 7–14 batches.     

Acid proteinases production by humicola lutea cells immobilized in polyhydroxyethylmethacrylate gel

The spores of Humicola lutea entrapped in polyhydroxyethylmethacrylate gel were precultivated in production medium for mycelial formation. The immobilized mycelium was reused in batch mode for acid proteinases production. The influence of precultivation time, initial inoculum gel volume, and gel particle size on the enzyme activity and proteinases production half-life were studied. After 70 h precultivation of the entrapped spores (10 ml initial inoculum volume, 12–27 mm³ gel particle size) maximum proteinases activity of 100–140% (compared with free cells) was registered in 15 reaction cycles. Under the same condition the half-life time was 18 cycles, while for the free cells it was 5 cycles. The main advantage of the polyhydroxyethylmethacylate immobilized H. lutea was the long acid proteinases production half-life at a low concentration of outgrowing cells in the medium.     

A sensitive polyacrylamide disc gel method for detection of proteinases

To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.     

Detection and characterization of epidermal proteinases by polyacrylamide gel electrophoresis

Electrophoresis of cornified cell extracts of 2-day-old rats, using SDS polyacrylamide gels copolymerized with alpha-casein or gelatin with or without plasminogen, was performed. Both Tris-HCl buffer soluble protein and KSCN solubilized protein contained a number of hydrolases for alpha-casein and/or gelatin, but PA (mol. wts 57 and 50K) was found only in the KSCN extract. The pH dependency, substrate specificity and mol. wt of plasminogen-independent proteinases were comparatively determined and DFP inhibition tested. This simple technique helped to identify the presence of several proteinases and to characterize them in partially purified epidermal extracts.     

Neuroendocrine control of moulting cycle in mealworms: Bioassay of moulting hormone

The moulting hormone content of mealworm homogenates was determined by injection of partially purified fractions into abdomens of mature larvae of Musca domestica. In mealworms with a 12-day interval between ecdyses, moulting hormone was at a maximum at 8 days.     

Messenger ribonucleoprotein particles in silkmoth oogenesis

Silkmoth oocytes contain significant amounts of nontranslating cytoplasmic messenger RNA apparently stored until fertilization. The physical state of this mRNA was examined by bouyant density centrifugation on cesium chloride gradients. Messenger elements were isolated either by oligo(dT) cellulose chromatography or by separation of ooplasm on sucrose gradients. After CsCl density gradient centifugation the mRNA particles banded in a region (1.42–1.48 g/c³) which would indicate a substantial protein content. Electron microscope examination of mRNP fractions revealed particles ranging in size from 180–250 Å.     

Inactive hepatic lipase in rat plasma

Hepatic lipase activity is detectable in liver but also in adrenal glands, ovaries, and plasma. The subunit size of hepatic lipase in liver, adrenal glands, and nonheparin plasma was compared. Hepatic lipase in liver and adrenal glands appeared as a 55 kDa band. In liver, a faint band of lower size was also detected. In nonheparin plasma, hepatic lipase appeared as a doublet of 57 kDa and 59 kDa. When activity/mass ratio was calculated, similar values were obtained for liver and adrenal glands. In plasma this value was much lower. After heparin administration in vivo, hepatic lipase activity in plasma increased nearly 100-fold with appearance of an additional 55 kDa band in postheparin plasma. This band coeluted with activity after preparative polyacrylamide gel electrophoresis. Differences in size persisted after digestion with peptide-N-glycosidase F. A progressive increase in 57 kDa and 59 kDa in postheparin plasma followed disappearance of the 55 kDa band, suggesting that these larger bands originate from the smaller form. In plasma, both smaller and larger forms were associated with HDL, but not with LDL or VLDL. We conclude that rat plasma contains a larger form of hepatic lipase that is inactive in in vitro assay.     

Degeneration of moulting glands in male crickets

The degeneration of the prothoracic glands of the male cricket, Gryllus bimaculatus, was analyzed by using an in vitro assay for ecdysteroid release from the moulting glands in last instar nymphs as well as in adult animals, and correlated with light and transmission electron microscopy. Apoptosis was examined by the TUNEL-reaction. The ability to synthesize ecdysteroids reached a peak at the 8th day of the last larval instar, identified as the moulting peak. After adult ecdysis it decreased to barely measurable values. Prothoracic gland degeneration was initiated at the time of the moulting peak, characterized by TUNEL positive reactions, nuclear and cytoplasmatic condensation, a striking abundance of residual basal laminae; besides a great amount of autophagic vacuoles are observed. The results reveal that apoptosis and autophagy are the basic mechanisms for programmed cell death in the prothoracic gland of Gryllus bimaculatus.     

The significance of moulting in Ecdysozoan evolution

SUMMARY Three major bilaterian clades first appear in the Early Cambrian fossil record: Deuterostomia, Lophotrochozoa, and Ecdysozoa. The taxa placed in Ecdysozoa are characterized by a moulting habit, unknown in the other major clades. The origin and consequences of moulting are of fundamental importance to the history of the ecdysozoan clade, chiefly because moulting precludes motile ectodermal cilia. Moulting may have originated as an adaptation to permit the enlargement, during growth, of secreted cuticular spines, flanges, and other structures used as ancillary locomotory devices. A combination of phylogenetic and fossil evidence suggests that the early members of these clades were small vermiform paracoelomates that likely lacked indirect-developing planktotrophic larvae. Thus, the evolution of planktotrophic larvae may have been independently achieved at least three times within Bilateria. The nonmoulting clades evolved larvae that swim and feed via ciliated tufts and bands, presumably intercalating these forms within their early developmental systems. Within Ecdysozoa, feeding larvae lacked ciliary feeding tracts and evolved by modification of early instars, employing limbs or setae to generate feeding currents. The setting aside during larval life of cells that give rise to adult features is probably an adaptation associated with metamorphosis.     

ATP-dependent proteinases in bacteria

Cytoplasmic proteolysis is an indispensable process for proper function of a cell. Degradation of many intracellular proteins is initiated by ATP-dependent proteinases, which are involved in the regulation of the level of proteins with short half-lives. In addition, they remove many damaged and abnormal proteins and thus play also an important role during stress. ATP-dependent proteinases are large multi-subunit assemblies composed of proteolytic core domains and ATPase-containing regulatory domains on a single polypeptide chain or on distinct subunits, which can act as molecular chaperones. This review briefly summarizes the data about four main groups of these proteinases in bacteria (i.e. Lon, Clp family, HsIUV and FtsH) and characrizes their structure, mechanism of action and properties.     

Cysteine proteinases in chondrosarcomas

The aim of the present study was to define the role of cathepsins B, H, K, L and S in the pathogenesis of human chondrosarcomas. For this purpose 40 tumour samples obtained from 12 patients with the diagnosis of conventional chondrosarcoma were systematically investigated for the expression of cathepsin mRNAs by Northern hybridisation, and for immunohistochemical localisation of the proteins. Northern analysis demonstrated the highest levels of cathepsins B and L in a recurring grade 1 chondrosarcoma, and in a grade 3 chondrosarcoma and in fibrous histiocytomas. Increased expression of cathepsin K mRNA was seen in seven chondrosarcomas, as well as in control tumours; fibrous histiocytomas, osteosarcomas, enchondromas and a giant cell tumour of bone. Cathepsin L was immunolocalised within the large chondrocytes, while cathepsin K was predominantly localised in large multinucleated osteoclastic cells and in some hypertrophic chondrocytes. These results suggest that chondrosarcoma can be included in the growing list of tumours, where cathepsins may well be involved in tumour progression. The simultaneous upregulation of cathepsins B and L, together with matrix metalloproteinase-13, and the association of cathepsin K with negative prognostic parameters suggests that an aggressive biological behaviour of chondrosarcoma may be related to the synthesis of cysteine proteinases and activation of other proteolytic enzymes. If this turns out to be the case, cathepsin inhibitors could provide the much needed adjuvant therapy for chondrosarcomas.     

The role of moulting in parasite defence

Parasitic infections consist of a succession of steps during which hosts and parasites interact in specific manners. At each step, hosts can use diverse defence mechanisms to counteract the parasite's attempts to invade and exploit them. Of these steps, the penetration of parasites into the host is a key step for a successful infection and the epithelium is the first line of host defence. The shedding of this protective layer (moulting) is a crucial feature in the life cycle of several invertebrate and vertebrate taxa, and is generally considered to make hosts vulnerable to parasites and predators. Here, we used the crustacean Daphnia magna to test whether moulting influences the likelihood of infection by the castrating bacterium Pasteuria ramosa. This parasite is known to attach to the host cuticula before penetrating into its body. We found that the likelihood of successful parasite infection is greatly reduced if the host moults within 12 h after parasite exposure. Thus, moulting is beneficial for the host being exposed to this parasite. We further show that exposure to the parasite does not induce hosts to moult earlier. We discuss the implications of our findings for host and parasite evolution and epidemiology.     

Stretch-induced moulting in Oncopeltus fasciatus

Fifth instar nymphs of Oncopeltus fasciatus initiate a moulting cycle when they reach a critical weight of 27.9±1.1 (±S.D.) mg for females or 23.7±1.2 mg for males. This critical weight is not absolute. Its precise value depends on the somatic size of the individual. Large-bodied individuals have a higher critical weight than small-bodied ones. Sexual size-dimorphism accounts largely for sex differences in critical weights. Animals of a subcritical weight can be induced to moult by means of a saline injection, in the absence of further food intake or growth. Since a saline injection only stretches the abdominal wall it is suggested that moulting in Oncopeltus is triggered by stimulation of abdominal stretch receptors, as in bloodsucking Reduviidae. The critical weight is thus an index of the size at which a critical degree of stretch is achieved.     

Heat-inducible transgenic expression in the silkmoth Bombyx mori

Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25 degrees C, 1 h at 42 degrees C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.     

Expression of giant silkmoth cecropin B genes in tobacco

Cecropin B is a small antibacterial peptide from the giant silkmothHyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco viaAgrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance toPseudomonas solanacearum, the causal agent of bacterial wilt of many crops, andP. syringae pv.tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin Bin vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.     

Amyloids protect the silkmoth oocyte and embryo

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of proteins that have remarkable mechanical and chemical properties protecting the oocyte and the developing embryo from a wide range of environmental hazards. We present data from electron microscopy (negative staining and shadowing), X-ray diffraction and modeling studies of synthetic peptide analogues of silkmoth chorion proteins indicating that chorion is a natural amyloid. The folding and self-assembly models of chorion peptides strongly support the beta-sheet helix model of amyloid fibrils proposed recently by Blake and Serpell [Structure 4 (1996) 989-998].     

X-ray diffraction studies of a silkmoth chorion

High- and low-angle diffraction studies have been performed on mature chorion (eggshell) of the silkmoth, Antheraea polyphemus. The results confirm the prevalence of β-sheet structure, previously suggested by predictions based on known primary structure and by results of laser Raman spectroscopy. The patterns obtained with different irradiation geometries suggest that a significant proportion of β-sheets are stacked and oriented with respect to the chorion surface and the ultrastructurally evident fibrillar components. Strong similarities are evident with the organization of β-sheets in chicken scale keratin.