Hydrolysis of fungal and plant cell walls by enzymatic complexes from cultures of Fusarium isolates with different aggressiveness to rye (Secale cereale)

The efficiency of hydrolysis of fungal (Fusarium spp.) cell wall and rye root cell wall by crude enzymatic complexes from (42-day-old) cultures of three F. culmorum isolates, a plant growth-promoting rhizosphere isolate (PGPF) DEMFc2, a deleterious rhizosphere isolate (DRMO) DEMFc5, and a pathogenic isolate DEMFc37, as well as two other, pathogenic isolates belonging to F. oxysporum and F. graminearum species was studied. In the enzymatic complexes originating from the Fusarium?spp. cultures, the activities of the following cell wall-degrading enzymes were identified: glucanases, chitinases, xylanases, endocellulases, exocellulases, pectinases, and polygalacturonases. The preparation originating from a culture of the PGPF isolate was the least efficient in plant cell wall (PCW) hydrolysis. There were no significant differences in the efficiency of PCW hydrolysis between preparations from cultures of the DRMO and the pathogenic isolates. PGPF was the most efficient in liberating reducing sugars and N-acetylglucosamine (GlcNAc) from fungal cell walls (FCW). Xylanase activities of the enzymatic complexes were strongly positively (R?>?+0.9) correlated with their efficiency in hydrolyzing PCW, whereas chitinase activities were correlated with the efficiency in FCW hydrolysis.     

Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

Background  

DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM).     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Increased prevalence of metabolic syndrome in patients with acne inversa

Background

Acne inversa (AI; also designated as Hidradenitis suppurativa) is a common chronic inflammatory skin disease, localized in the axillary, inguinal and perianal skin areas that causes painful, fistulating sinuses with malodorous purulence and scars. Several chronic inflammatory diseases are associated with the metabolic syndrome and its consequences including arteriosclerosis, coronary heart disease, myocardial infraction, and stroke. So far, the association of AI with systemic metabolic alterations is largely unexplored.

Methods and Findings

A hospital-based case-control study in 80 AI patients and 100 age- and sex-matched control participants was carried out. The prevalence of central obesity (odds ratio 5.88), hypertriglyceridemia (odds ratio 2.24), hypo-HDL-cholesterolemia (odds ratio 4.56), and hyperglycemia (odds ratio 4.09) in AI patients was significantly higher than in controls. Furthermore, the metabolic syndrome, previously defined as the presence of at least three of the five alterations listed above, was more common in those patients compared to controls (40.0% versus 13.0%; odds ratio 4.46, 95% confidence interval 2.02 to 9.96; P<0.001). AI patients with metabolic syndrome also had more pronounced metabolic alterations than controls with metabolic syndrome. Interestingly, there was no correlation between the severity or duration of the disease and the levels of respective parameters or the number of criteria defining the metabolic syndrome. Rather, the metabolic syndrome was observed in a disproportionately high percentage of young AI patients.

Conclusions

This study shows for the first time that AI patients have a high prevalence of the metabolic syndrome and all of its criteria. It further suggests that the inflammation present in AI patients does not have a major impact on the development of metabolic alterations. Instead, evidence is given for a role of metabolic alterations in the development of AI. We recommend monitoring of AI patients in order to correct their modifiable cardiovascular risk factors.     

Ceramide profiles in the brain of rats with diabetes induced by streptozotocin

Diabetes is associated with disturbances of brain activity and cognitive impairment. We hypothesize that ceramides may constitute an important contribution to diabetes-linked neuro-dysfunction. In our study we used rats injected with streptozotocin (STZ) as a model of severe hyperglycemia. Using the gas-liquid chromatography technique we found a significant increase of ceramide content in brains and a decrease in plasma of diabetic rats. The inhibitor of serine palmitoyltransferase, myriocin, reduced ceramide generation in hyperglycemic brains, although injected alone it exerted a paradoxical effect of ceramide upregulation. Myriocin had no impact on ceramide concentration in the plasma of either control or diabetic rats. The level of ceramide saturated fatty acids was elevated whereas the level of ceramide poly-unsaturated fatty acids was downregulated in brains of all experimental groups. The concentration of ceramide mono-unsaturated fatty acids remained unchanged. The pattern of individual ceramide species was altered depending on treatment. We noted an STZ-evoked increase of brain ceramide C16:0, C18:0 and C20:0 and a strong decline in ceramide C18:2 fatty acid levels. Some changes of brain ceramide pattern were modified by myriocin. We found a decreased amount of total ceramide-ω-6 fatty acids in STZ-treated rat brains and no changes in ceramide-ω-3 concentration. We conclude that ceramides may be important mediators of diabetes-accompanied brain dysfunction.     

Determination of fluorescent probes localization in membranes by nonradiative energy transfer

One of the new methods of studying the structure and dimensions of biological membranes is based on the F?rster's nonradiative energy transfer between special molecules, the so-called 'membrane fluorescent probes'. Further development of the approach is presented in this article. It consists of the combined use of the time-resolved and steady-state fluorescence data with subsequent computer simulation of the energy transfer in membranes. Anthracene as an energy donor, and 4-p-(dimethylamino)styryl-N-dodecylpyridinium (DSP-12) or 4-dimethylaminochalcone (DMC) as energy acceptors were bound with artificial phospholipid membrane vesicles ('liposomes'). The synchrotron radiation was used as an impulse source for the excitation light. The steady-state fluorescence data permit the area of possible probe localization in membranes to be distinguished, while the kinetic data allow them to be narrowed significantly. There is a good agreement between the obtained localization and our present-day knowledge of lipid bilayer structure. The accuracy of the method is ca. several Angstr?ms.     

Replication of DNA in mammalian chromosomes

DNA replication has been studied in cells (CHO) synchronized by mitotic selection from roller cultures. A study of the incorporation of ³H supplied as uridine indicates that cells cannot be blocked precisely at the beginning of the S phase, but DNA synthesis can be stopped in early S by treating with F-dU in G₁. After blockage potential initiation sites continue to increase at a linear rate for atleast 13 hours after division. Incorporation of ³H-thymidine begins at most of these sites within seconds after thymidine is supplied in the medium and incorporation continues at a linear rate for 20–24 minutes. There appears to be a pause after this interval before synthesis is resumed at about two times the initial rate. ³H-bromodeoxyuridine can be substituted for thymidine without affecting the kinetic pattern over a similar period. The increased rate is probably an increase in sites of chain growth rather than a change in rate of chain growth. A study of the labeled DNA segments by band sedimentation in a preformed NaClO₄ isokinetic gradient shows that two distinctly different sized segments can be released from the chromosomes by lysis at submelting conditions. One is the previously reported single chain segments averaging about one-half micron in length, but the other is a much larger segment (26S) which is native DNA with perhaps small regions of single chains presumably at the ends. Primarily single chain DNA is released after 1–2 minute pulse labeling, but after 2 minutes the larger segments (26S) contain most of the newly formed DNA except that attached to the chains of the major part of the template DNA which exhibits a discontinuous distribution, sedimenting far faster than either newly replicated segment. A consideration of the kinetics of formation of the 26S component indicates that is may contain the replicating fork. If this proves to be the correct interpretation the template chains would both have non-adjacent nicks preceeding the fork and also in a post-fork site at a mean distance of about 2 microns in both directions. The isolation of the growing points of DNA replication in chromosomes is now possible and the study of properties of the newly replicated regions should be greatly facilitated.     

Patterns of population genetic variation in sympatric chiltoniid amphipods within a calcrete aquifer reveal a dynamic subterranean environment

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km² region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.     

Prospects of application of the chitin-binding domains to isolation and purification of recombinant proteins by affinity chromatography

Properties of substrate-binding domains, some parameters of affinity sorbents, and a number of other special features that were necessary to take into account during creation of chromatographic system for isolation and purification of proteins with incorporated chitin-binding domain were discussed in this review. This method was shown to be successfully used along with metal-chelate affinity chromatography. The metal-chelate affinity chromatography with the use of polyhistidine peptides as affinity labels is successfully applied to isolation, purification, and investigation of recombinant proteins. However, this system had some disadvantages. At present, scientists attracted more and more attention to substrate-binding domains, including those chitin-binding, because they had a number of advantages being used as affinity label.     

Size classes of replication units in DNA from sea urchin embryos

Sea urchin DNA containing replication structures was isolated from two to four cell stage and blastula stage embryos, and examined by electron microscopy. In addition to the expected eye forms, we also observed molecules with large internal single-stranded gaps. Such structures were not present in DNA devoid of replicating molecules such as that isolated from sea urchin sperm. When the size of eye forms and interbubble distances between the two stages were compared, there was no detectable difference. In both stages, we observed two distinct size classes of bubbles and of interbubble distances. In the case of bubble sizes, the smaller size class was comprised of clustered microbubbles that ranged from 200 base pairs to 1 Kilobase (kb) with a mean of 432 base pairs. The large eye forms measured 1--35 kb with a mean of 6.8 kb. Interbubble distances also yielded two distinct populations, with the smaller class ranging from 400 base pairs to 2.3 kb (mean = 1.1 kb) and the larger population ranging from 2.8 to 36 kb (mean = 10.9 kb). Although other possibilities cannot be entirely excluded, the data support the contention that a substantial fraction of the larger eye-form population arises from the fusion of the clustered microbubbles.