An Ihproved Purification Method for Cytosolic Malate Dehydrogenase from Several Sources

A new purification method for cytosolic ma late dehydrogenases from several sources has been developed. The procedure, employing chromatographies on 5′AMP-Sepharose, DEAE-Sephacel and Blue-Sepharose, allows for a rapid isolation of the enzyme (% 40 hours), in large quantities, with good yields (45–54 %). The specific activity of final preparations were around 1300 I. U. /mg and were judged homogeneous by polyacrylamide gradient gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size exclusion chromatography and isoelectric focusing.     

Heterozoan carbonate-enriched beach sand and coastal dunes—with particular reference to rhodoliths,Dirk Hartog Island,Shark Bay,Western Australia

This is a preliminary interdisciplinary study on the enrichment of heterozoan carbonates on Dirk Hartog Island, Shark Bay, Western Australia, with particular reference to rhodolith (free-living non-geniculate) coralline algae. The current study aims to investigate the geological impact of shallow-water rhodoliths in Shark Bay, as well as fill critical information gaps on the biogeographical distribution of rhodoliths in Australia. We analyzed the composition of sand from eight sites (totaling 21 beach and sand dune samples) on the eastern (windward) shore of the island, and investigated the origin of the coralline algal grains. Heterozoan carbonates (shell, geniculate coralline algae grains, and rhodolith grains) together comprised 3–84% of the carbonate-enriched beach and dune sand samples. While shell fragments often comprised the highest percentage (up to 73%), rhodolith grains (up to 27%) were found in 12 of 21 samples, with rhodolith grains also occurring in two dune samples. Geologically, the study has shown that rhodoliths and rhodolith beds are important shallow-marine habitats in Shark Bay, with a proven capacity to enrich beach/dune sands in Shark Bay and potentially other areas along the Australian coast. Biogeographically, the study confirmed the presence of a previously undescribed shallow rhodolith bed in Shark Bay (the first bed documented on the Western shore) with the possibility of a third bed near Sandy Point on Dirk Hartog Island. It also confirmed the presence of rhodolith forming Neogoniolithon brassica-florida and Lithophyllum sp. in Shark Bay, and is the first record of Hydrolithon reinboldii rhodoliths in Australia.     

Role of Amyloid Precursor Protein (APP) and Its Derivatives in the Biology and Cell Fate Specification of Neural Stem Cells

Amyloid precursor protein (APP) is a member of the APP family of proteins, and different enzymatic processing leads to the production of several derivatives that are shown to have distinct biological functions. APP is involved in the pathology of Alzheimer’s disease (AD), the most common neurodegenerative disorder causing dementia. Furthermore, it is believed that individuals with Down syndrome (DS) have increased APP expression, due to an extra copy of chromosome 21 (Hsa21), that contains the gene for APP. Nevertheless, the physiological function of APP remains unclear. It is known that APP plays an important role in neural growth and maturation during brain development, possibly by influencing proliferation, cell fate specification and neurogenesis of neural stem cells (NSCs). Proteolytic cleavage of APP occurs mainly via two mutually exclusive pathways, the non-amyloidogenic pathway or the amyloidogenic pathway. Other alternative pathways (η-secretase, δ-secretase and meprin pathways) have also been described for the physiological processing of APP. The different metabolites generated from these pathways, including soluble APPα (sAPPα), soluble APPβ (sAPPβ), β-amyloid (Aβ) peptides and the APP intracellular domain (AICD), have different functions determined by their structural differences, equilibrium and concentration with respect to other fragments derived from APP. This review discusses recent observations regarding possible functions of APP and its proteolytic derivatives in the biology and phenotypic specification of NSCs. This can be important for a better understanding of the pathogenesis and the development of future therapeutic applications for AD and/or DS, diseases in which alterations in neurogenesis have been described.     

Do whiskers contribute to nipple-search and suckling behavior in newborn rabbits (Oryctolagus cuniculus)?

Rabbit pups are only nursed for about 3 min once a day. They depend on a pheromone on the mother's ventrum to locate nipples and on tactile stimulation of the muzzle to grasp them. In a continuing study of the sensory input guiding suckling behavior we investigated the whisker array in newborn pups and the possible contribution of the whiskers to suckling. Rabbits are born with approximately 76 whiskers arranged in seven to nine rows and increasing in length from rostral to caudal. No significant difference was found between pups with whiskers cut and intact controls in latency to perform the stereotyped nipple-search behavior, latency to attach to nipples, time spent on nipples, milk ingested, or in the strength of conditioning to a novel odor paired with suckling. Thus, the whiskers do not seem important for suckling in newborn rabbits.

Zusammenfassung

Tragen die Schnurrhaare bei neugeborenen Kaninchen (Oryctolgus cuniculus) zum Zitzensuch- und Saugverhalten bei?Jungkaninchen werden nur einmal am Tag für etwa drei Minuten gesäugt. Für das Auffinden der Zitzen sind sie auf ein Pheromon auf der Bauchhaut der Mutter angewiesen, und für deren Ergreifen auf periorale taktile Reize. In Fortsetzung unserer Untersuchungen zur sensorischen Kontrolle dieses Verhaltens beschreiben wir die Anordnung der Schnurrhaare bei neugeborenen Kaninchen und untersuchen ihren möglichen Beitrag zum Säugeverhalten. Bei der Geburt besitzen Jungkaninchen etwa 76, in 7–9 Reihen angeordnete Schnurrhaare, deren Länge von rostral nach caudal zunimmt. Nach Abschneiden der Schnurrhaare wurden keine signifikanten Unterschiede zwischen Jungen mit und ohne Schnurrhaare beobachtet, sowohl in der Latenz des Zitzensuchens, des Zitzenfassens, wie in der Besaugungsdauer, der erhaltenen Milchmenge oder dem Grad der Konditionierung auf einen neuen Geruchsstoff. Demnach spielen bei neugeborenen Kaninchen die Schnurrhaare keine wesentliche Rolle für das Auffinden und Besaugen von Zitzen.     

Implementation of intensity modulated radiotherapy for prostate cancer in a private radiotherapy service in Mexico

Intensity modulated radiation therapy (IMRT) allows physicians to deliver higher conformal doses to the tumour, while avoiding adjacent structures. As a result the probability of tumour control is higher and toxicity may be reduced. However, implementation of IMRT is highly complex and requires a rigorous quality assurance (QA) program both before and during treatment. The present article describes the process of implementing IMRT for localized prostate cancer in a radiation therapy department. In our experience, IMRT implementation requires careful planning due to the need to simultaneously implement specialized software, multifaceted QA programs, and training of the multidisciplinary team. Establishing standardized protocols and ensuring close collaboration between a multidisciplinary team is challenging but essential.     

Examination of the genetic variability among biofilm-forming Candida albicans clinical isolates

Biofilms are microbial communities encased in a self-produced polymeric matrix and represent a common mode of microbial growth. Candida albicans is able to colonize the surface of catheters, prostheses, and epithelia, forming biofilms that are highly resistant to antimicrobial drugs. The objective of this study was the genotypic characterization of biofilm-forming C. albicans clinical isolates using RAPD (Random Amplified Polymorphic DNA). We have studied 25 clinical isolates of C. albicans from oral cavities, blood, skin, nail, stool, oesophagus biopsy and vaginal fluids from patients suffering from candidiasis. For each strain biofilm formation was analysed by measuring the ability to adhere to and grow on polystyrene plastic surfaces using XTT [2,3-bis(2-methoxi-4nitro-5sulfophenil)-2H tetrazolium-5carboxanilide] reduction assay. The similarity coefficients generated by RAPD using four different primers varied from 49 to 91%, indicating a high degree of genetic variability between the clinical isolates. The dendrogram clustered the isolates in four related groups, all groups included strains with very different abilities to form biofilms. The isolates with similar genotypes often showed very different biofilm formation abilities. Strains were grouped into clusters independently of their clinical sources. Our results suggested that a direct correlation does not exist between the biofilm-forming ability of natural populations of C. albicans and the genotype as determined by RAPD.     

Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: a model system

Ticks (Acari: Ixodidae) are ubiquitous hosts of rickettsiae (Rickettsiaceae: Rickettsia), obligate intracellular bacteria that occur as a continuum from nonpathogenic arthropod endosymbionts to virulent pathogens of both arthropod vectors and vertebrates. Visualization of rickettsiae in hosts has traditionally been limited to techniques utilizing fixed tissues. We report epifluorescence microscopy observations of unfixed tick tissues infected with a spotted fever group endosymbiont, Rickettsia monacensis, transformed to express green fluorescent protein (GFP). Fluorescent rickettsiae were readily visualized in tick tissues. In adult female, but not male, Ixodes scapularis infected by capillary feeding, R. monacensis disseminated from the gut and infected the salivary glands that are crucial to the role of ticks as vectors. The rickettsiae infected the respiratory tracheal system, a potential dissemination pathway and possible infection reservoir during tick molting. R. monacensis disseminated from the gut of capillary fed I. scapularis nymphs and was transstadially transmitted to adults. Larvae, infected by immersion, transstadially transmitted the rickettsiae to nymphs. Infected female I. scapularis did not transovarially transmit R. monacensis to progeny and the rickettsiae were not horizontally transmitted to a rabbit or hamsters. Survival of infected nymphal and adult I. scapularis did not differ from that of uninfected control ticks. R. monacensis did not disseminate from the gut of capillary fed adult female Amblyomma americanum (L.), or adult Dermacentor variabilis (Say) ticks of either sex. Infection of I. scapularis with R. monacensis expressing GFP provides a model system allowing visualization and study of live rickettsiae in unfixed tissues of an arthropod host.     

An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn²⁺ bound (PDB_ID:4PCA), and SAM- Mn²⁺ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.     

Phylogenetic Relationships of Butyrate-Producing Bacteria from the Human Gut

Butyrate is a preferred energy source for colonic epithelial cells and is thought to play an important role in maintaining colonic health in humans. In order to investigate the diversity and stability of butyrate-producing organisms of the colonic flora, anaerobic butyrate-producing bacteria were isolated from freshly voided human fecal samples from three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. A second isolation was performed on the same three individuals 1 year later. Of a total of 313 bacterial isolates, 74 produced more than 2 mM butyrate in vitro. Butyrate-producing isolates were grouped by 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. The results indicate very little overlap between the predominant ribotypes of the three subjects; furthermore, the flora of each individual changed significantly between the two isolations. Complete sequences of 16S rDNAs were determined for 24 representative strains and subjected to phylogenetic analysis. Eighty percent of the butyrate-producing isolates fell within the XIVa cluster of gram-positive bacteria as defined by M. D. Collins et al. (Int. J. Syst. Bacteriol. 44:812–826, 1994) and A. Willems et al. (Int. J. Syst. Bacteriol. 46:195–199, 1996), with the most abundant group (10 of 24 or 42%) clustering with Eubacterium rectale, Eubacterium ramulus, and Roseburia cecicola. Fifty percent of the butyrate-producing isolates were net acetate consumers during growth, suggesting that they employ the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. In contrast, only 1% of the 239 non-butyrate-producing isolates consumed acetate.