β-Linked disaccharides stimulate,but do not act as primer for,β(1–3)glucan synthase activity ofNeurospora crassa

-Linked disaccharides (laminaribiose and cellobiose) stimulated(1–3)glucan synthase activity ofNeurospora crassa by reducing the K_{m app} for the substrate while not changing the V_max. Laminaribiose and cellobiose werelinear activators with a K_{a app} of 0.32 mM and K_{a app} of 1.7 mM, respectively. Laminaribiose was not found to be incorporated into product, i.e., did not act as a primer covalently bound to product.     

Permeabilization ofNeurospora crassa hyphae with toluene-ethanol and filipin

Young hyphae ofNeurospora crassa were made permeable to UDP-glucose and trypan blue by treatment with toluene-ethanol and filipin. Less than 2% of treated cells survived treatment with 8% and 16% toluene-ethanol, while 25% survived treatment with 4% toluene-ethanol. Similarly, 98% of treated cells were killed by treatment with 16 g/ml filipin. Electron microscopy revealed that toluene-ethanol-treated cells lost pieces of plasma membrane and contained a number of vacuole-like structures; filipin-treated cells were less affected. Both filipin- and toluene-ethanol-treated cells were able to incorporate UDP-glucose into insoluble material (likely glycogen and glucan).     

(1–3)-β-Glucan synthesis ofNeurospora crassa

(1–3)--d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

Chitin synthetase ofNeurospora crassa: Inhibition by nikkomycin,polyoxin B,and UDP

The inhibitory effects of nikkomycin, polyoxin B, and UDP were tested on particulate chitin synthetase activity (UDP-2-acetamido-2-deoxy-D-glucose: chitin-4-B-acetamidodeoxy-D-glucosyltransferase, E.C.2.4.1.16) fromNeurospora crassa. Two approaches were used: (a) inhibitors were tested for their individual effects on chitin synthetase activity; (b) paired combinations of inhibitors were examined to establish whether the compounds affected inhibition by binding at the same enzyme site. The first method showed that the three compounds are linear competitive inhibitors, i.e. each competes directly with the substrate for binding. K_{i app} values were: UDP, 0.8 mM; polyoxin B, 32 M; and nikkomycin, 2 M. The second method showed that the inhibitors compete with each other for binding; thus they bind at the same site. The pyrimidine nucleoside moiety of these inhibitors is an essential component for effective inhibition, but the potency of inhibition is critically dependent on the conformation of the side group attached to carbon 5 of the ribose sugar.     

Formation and regeneration of protoplasts of wild-typeNeurospora crassa

Trichoderma reesei was grown using purified cell walls ofNeurospora crassa as a primary source of carbon. The resulting culture medium contained an undefined mixture ofN. crassa cell-wall digesting enzymes. Protoplasts (cell lacking wall) were formed when youngN. crassa hyphae were treated withTrichoderma mixture. The vast majority of protoplasts resynthesized cell-wall material when washed free of cell-wall digesting enzyme; of these, about 40% regenerated a mycelium.     

Zeamatin inhibits trypsin and alpha-amylase activities

Zeamatin is a 22-kDa protein isolated from Zea mays that has antifungal activity against human and plant pathogens. Unlike other pathogenesis-related group 5 proteins, zeamatin inhibits insect alpha-amylase and mammalian trypsin activities. It is of clinical significance that zeamatin did not inhibit human alpha-amylase activity and inhibited mammalian trypsin activity only at high molar concentrations.