Yeast cells of Candida albicans are induced by serum at 37 6C to produce germ tubes, the first step in a transition from yeast to hyphal growth. Previously, it has been shown that the active component is not serum albumin but is present in the dialysable fraction of serum. In this study, serum induction of germ-tube formation is shown to occur even in the presence of added exogenous nitrogen sources and is therefore not signalled by nitrogen derepression. The active component in serum was purified by ion-exchange, reverse-phase and size-exclusion chromatography from the dialysable fraction of serum and was identified by NMR to be D-glucose. Enzymic destruction of glucose, using glucose oxidase, demonstrated that D-glucose was the only active component in these fractions. Induction of germ-tube formation by D-glucose required a temperature of 37 6C and the pH optimum was between pH 7?0 and 8?0. D-Glucose induced germ-tube formation in a panel of clinical isolates of C. albicans. Although D-glucose is the major inducer in serum, a second non-dialysable, trichloroacetic acid precipitable inducer is also present. However, whereas either 1?4 % (v/v) serum or an equivalent concentration of D-glucose induced 50 % germ-tube formation, the non-dialysable component required a 10-fold higher concentration to induce 50 % germ-tube formation. Serum is, therefore, the most effective induction medium for germ-tube formation because it is buffered at about pH 8?5 and contains two distinct inducers (glucose and a non-dialysable component), both active at this pH.

Phloem exudate from squash fruit contains heat-inactivated material which inhibits pepsin activity. This inhibitory activity was purified by mild acid treatment, chromatography on trypsin-agarose, Sephadex G-75 and reverse-phase HPLC, resulting in the elution of three peaks with pepsin-inhibitory activity. N-terminal sequencing indicated a common sequence of MGPGPAIGEVIG and the presence of minor species with seven-or two-amino-acid N-terminal extensions beyond this point. Microheterogeneity in this end sequence was exhibited within and between two preparations. Internal sequencing of a major peak after a trypsin digestion gave the sequence FYNVVVLEK. The common N-terminal sequence was used to design a degenerate primer for 3′ rapid amplification of cDNA ends and cDNA clones encoding two isoforms of the inhibitor were obtained. The open reading frames of both cDNAs encoded proteins (96% identical) which contained the experimentally determined internal sequence. The amino acid content calculated from the predicted amino acid sequence was very similar to that measured by amino acid analysis of the purified inhibitor. The two predicted amino acid sequences (96 residues) had neither similarity to any other aspartic proteinase inhibitor nor similarity to any other protein. The inhibitors have a molecular mass of 10 552 Da, measured by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and approximately 10000 Da by SDS/PAGE, and behave as dimers of approximately 21 000 Da during chromatography on Superdex G-75 gel-filtration medium. The calculated molecular masses from the predicted amino acid sequences were 10551 Da and 10527 Da. The inhibitor was capable of inhibiting pepsin (K i ϭ 2 nM) and a secreted aspartic proteinase from the fungus Glomerella cingulata (K i ϭ 20 nM). The inhibitor, which is stable over acid and neutral pH, has been named squash aspartic proteinase inhibitor (SQAPI).Keywords : aspartic-protease inhibitor ; pepsin; phloem; squash.Aspartic proteinases are found in a wide variety of organ-range of chemical approaches have been employed in designing new inhibitors, invariably synthetic approaches have mimicked isms, including viruses, fungi, plants and animals, where they carry out a diverse array of functions involving specific proteol-pepstatin in the provision of a gem diol (or analogue) of the key reaction intermediate [2Ϫ5]. ysis or more general degradation [1]. Well known examples include HIV protease, pepsin, chymosin, renin, and cathepsins D Aspartic proteinase inhibitors, that are themselves proteins, are scarce in nature. Only four sources of proteinaceous aspartic and E. These enzymes are characterized by their catalytic activity being dependent on a pair of aspartic acid residues in the proteinase inhibitor have been reported ; from potato [6], wheat [7], yeast [8] and the nematode (Ascaris spp.) [9]. The best charactive site and by generally operating at low pH. Most are inhibited by pepstatin, a non-proteinaceous microbial hexapeptide acterized of these, the...

Glomerella cingulata, which infects a number of different hosts, gains entry to the plant tissue by means of an appressorium. Turgor pressure generated within the appressorium forces a penetration peg through the plant cuticle. A visible lesion forms as the fungus continues to grow within the host. A G. cingulata homolog (GcSTUA) of the genes encoding Asm1, Phd1, Sok2, Efg1, and StuA transcription factors in Magnaporthe grisea and other fungi was cloned and shown to be required for infection of intact apple fruit and penetration of onion epidermal cells. Mobilization of glycogen and triacylglycerol during formation of appressoria by the GcSTUA deletion mutant appeared normal and melanization of the maturing appressoria was also indistinguishable from that of the wild type. However, GcSTUA was essential for the generation of normal turgor pressure within the appressorium. As is the case for its homologs in other fungi, GcSTUA also was required for the formation of aerial hyphae, efficient conidiation, and the formation of perithecia (sexual reproductive structures).

Objectives The induction of analgesia for many chronic cutaneous lesions requires treatment with an opioid analgesic. In many patients suffering with these wounds such drugs are either contraindicated or shunned because of their association with death. There are now case reports involving over 100 patients with many different types of chronic superficial wounds, which suggest that the topical application of an opioid in a suitable gel leads to a significant reduction in the level of perceived pain. Key findings Some work has been undertaken to elucidate the mechanisms by which such a reduction is achieved. To date there have been no proven deleterious effects of such an analgesic system upon wound healing. Although morphine is not absorbed through the intact epidermis, an open wound provides no such barrier and for large wounds drug absorption can be problematic. However, for most chronic cutaneous lesions, where data has been gathered, the blood levels of the drug applied ranges from undetectable to below that required for a systemic effect. Summary If proven, the use of opioids in this way would provide adequate analgesia for a collection of wounds, which are difficult to treat in patients who are often vulnerable. Proof of this concept is now urgently required.

Secretion of the extracellular Rhizopus carboxyl proteinase (EC 3.4.23.6) by Rhizopus oligosporus is repressed in the presence of low-molecular-mass sources of nitrogen, sulphur and carbon. Proteinase is secreted when the medium is deficient in any one of these three nutrients. In the case of nitrogen metabolite repression, control is at the level of transcription. Induction of proteinase secretion by exogenous protein does not occur in any of the media examined.