Modification of polysaccharides and plant cell wall by endo-1,4-beta-glucanase and cellulose-binding domains

Cellulose is one of the most abundant polymers in nature. Different living systems evolved simultaneously, using structurally similar proteins to synthesize and metabolize polysaccharides. In the growing plant, cell wall loosening, together with cellulose biosynthesis, enables turgor-driven cell expansion. It has been postulated that endo-1,4-beta-glucanases (EGases) play a central role in these complex activities. Similarly, microorganisms use a consortium of lytic enzymes to convert cellulose into soluble sugars. Most, if not all, cellulases have a modular structure with two or more separate independent functional domains. Binding to cellulose is mediated by a cellulose-binding domain (CBD), whereas the catalytic domain mediates hydrolysis. Today, EGases and CBDs are known to exist in a wide range of species and it is evident that both possess immense potential in modifying polysaccharide materials in-vivo and in-vitro. The hydrolytic function is utilized for polysaccharide degradation in microbial systems and cell wall biogenesis in plants. The CBDs exerts activity that can be utilized for effective degradation of crystalline cellulose, plant cell wall relaxation, expansion and cell wall biosynthesis. Applications range from modulating the architecture of individual cells to an entire organism. These genes, when expressed under specific promoters and appropriate trafficking signals can be used to alter the nutritional value and texture of agricultural crop and their final products. EGases and CBDs may also find applications in the modification of physical and chemical properties of composite materials to create new materials possessing improved properties.     

A Monte Carlo simulation of the determination of mean particle volume using the Cavalieri estimator

BACKGROUND: A common morphometric problem is the determination of an estimate of the size of biological particles obtained from measurements made on a sample of profiles observed in sections. Results are reported typically in terms of mean caliper diameter or mean volume of the particle. METHODS: We have investigated the use of the Cavalieri estimator for obtaining estimates of mean particle volume using a Monte Carlo simulation. Samples of spherical and ellipsoidal particles were generated by computer and serially sectioned at a fixed mean thickness with a small, imposed random variation. The area of each profile was determined and the volume of the particle was calculated according to the Cavalieri estimator. The influence on the estimate of the mean particle volume and its 95% confidence interval was evaluated for several variables: the shape of the particles, the standard deviation of the particle volume in the population, the section thickness, and the standard deviation of the section thickness. RESULTS: The results obtained with the Cavalieri estimator correspond favorably with those obtained with previously reported alternative methods. This leads to a recommendation for the strong consideration for the use of the Cavalieri estimator in cases in which it is technically feasible to obtain at least three sections through the individual particles. Graphs are provided, which relate the confidence interval for the mean volume to the number of particles measured.     

Development of Hepatozoon caimani (Carini, 1909) Pess a,De Biasi & De Souza, 1972 in the Caiman Caiman c. crocodilus,the frog Rana catesbeiana and the mosquito Culex fatigans

The sporogony of Hepatozoon caimani has been studied, by light microscopy, in the mosquito Culex fatigans fed on specimens of the caiman Caiman c. crocodilus showing gametocytes in their peripheral blood. Sporonts iniciate development in the space between the epithelium of the insect gut and the elastic membrane covering the haemocoele surface of the stomach. Sporulating oocysts are clustered on the gut, still invested by the gut surface membrane. Fully mature oocysts were first seen 21 days after the blood-meal. No sporogonic stages were found in some unidentified leeches fed on an infected caiman, up to 30 days following the blood-meal. When mosquitoes containing mature oocysts were fed to frogs (Leptodactylus fuscus and Rana catesbeiana), cysts containing cystozoites developed in the internal organs, principally the liver. Feeding these frogs to farm-bred caimans resulted in the appearance of gametocytes in their peripheral blood at some time between 59 and 79 days later, and the development of tissue cysts in the liver, spleen, lungs and kidneys. Transmission of the parasite was also obtained by feeding young caimans with infected mosquitoes and it is suggested that both methods occur in nature. The finding of similar cysts containing cystozoites in the semi-aquatic lizard Neusticurus bicarinatus, experimentally fed with infected C. fatigans, suggests that other secondary hosts may be involved.     

EspH,a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions. These include transient filopodium formation which is Cdc42-dependent. Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42. We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH. We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies. Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation. Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation. We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals. Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH. EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure. Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.     

Opening and closing of KCNKO potassium leak channels is tightly regulated

Potassium-selective leak channels control neuromuscular function through effects on membrane excitability. Nonetheless, their existence as independent molecular entities was established only recently with the cloning of KCNKO from Drosophila melanogaster. Here, the operating mechanism of these 2 P domain leak channels is delineated. Single KCNKO channels switch between two long-lived states (one open and one closed) in a tenaciously regulated fashion. Activation can increase the open probability to approximately 1, and inhibition can reduce it to approximately 0.05. Gating is dictated by a 700-residue carboxy-terminal tail that controls the closed state dwell time but does not form a channel gate; its deletion (to produce a 300-residue subunit with two P domains and four transmembrane segments) yields unregulated leak channels that enter, but do not maintain, the closed state. The tail integrates simultaneous input from multiple regulatory pathways acting via protein kinases C, A, and G.     

Changes along the pituitary-gonadal axis during maturation of the black carp, Mylopharyngodon piceus

The black carp, Mylopharyngodon piceus, is a late-maturing cyprinid reaching sexual maturity at the age of 6-7 years. The present work attempted to define nonfunctional sites along the pituitary-gonadal axis in immature fish utilizing in vivo and in vitro challenge experiments. Two- and 3-year old fish injected with salmon gonadotropin-releasing hormone analog (sGnRHa; 10 microg/kg) and metoclopramide (20 mg/kg) did not reveal any increase in circulating gonadotropin (cGtH) or estradiol (E(2)) level. Furthermore, cGtH release from cultured pituitary cells of fish at these ages did not increase in response to sGnRH (0.1 nM - 1 microM) but was augmented when exposed to TPA (12.5 nM). However, 4-year old female fish did respond to the above treatments both in vivo and in vitro. These results suggest the existence of nonfunctional site(s) proximal to the activation of PKC in the immature black carp gonadotrophs, probably at the level of GnRH receptors. These site(s) start to become functional in 4-year old fish. Two- and 3-year old fish injected with common carp pituitary extract (CPE) containing 350 microg cGtH/kg did not show any increase in circulating E(2). In addition, the estrogen secretion from fragments of the rudimentary gonads did not increase after exposure to CPE containing cGtH (0.5-4 microg/ml) but was elevated dose-dependently by exposure to dbcAMP (0.3-3 mM). However, the ovaries of 4-year old fish did respond to the gonadotropic stimulation, both in vivo and in vitro. These results suggest the existence of other non-functional site(s) in the immature black carp, proximal to the formation of cAMP in the gonads, probably at the level of GtH receptors. These site(s) start to become functional in 4-year old females. Another source of E(2) was discovered in the immature black carp: namely, the fat pad adjacent to the gonads. In contrast to the visceral adipose tissue, the fat pad secretes estrogen in response to cAMP elevation in 2- and 3-year old fish while in 4-year old fish it also responds to gonadotropic stimulation. Due to its large mass and high steroidogenic potency, it is assumed that the gonadal fat pad is involved in the process of puberty in the black carp. J. Exp. Zool. 286:405-413, 2000.