Mutagen effects on rat seminiferous tubules in vitro: induction of meiotic micronuclei by adriamycin

Mutagen effect on male germ cells can be analyzed by micronucleus induction during meiotic divisions. These can be followed in vitro by culturing seminiferous tubular segments from stages of the epithelial cycle that contain late pachytene and diakinetic primary spermatocytes. We studied the formation of micronuclei in this test system using adriamycin as a model mutagen. Micronuclei were induced in a dose-dependent manner at concentrations of 1-10 ng/ml that were far below the dose that caused morphologically or biochemically detectable cytotoxic effects. The meiotic micronucleus induction in vitro is a potentially sensitive test system of male germ cell mutagenesis.     

Identification of stage-specific proteins synthesized by rat seminiferous tubules

Experiments were conducted to determine how the cycle of the seminiferous epithelium influenced synthesis and secretion of proteins by seminiferous tubules. Tubular segments were treated with collagenase and then cultured with [35S]methionine. These myoid cell-depleted tubules isolated from different stages of the epithelial cycle exhibited, at Stages VI and XII, two distinct peaks of secretion of total radiolabeled proteins. Two-dimensional gel electrophoresis indicated that the patterns of secreted proteins from these two stages were remarkably different, while those from other stages were intermediate between those at the peaks. At least 15 proteins were secreted cyclically, many of them previously unrecognized products of the seminiferous epithelium. One product, designated Cyclic Protein-2 (CP-2), exhibited a pronounced cycle of secretion, its peak at Stage VI being 30-fold greater than at its nadir at Stages XII-XIV. Further investigation indicated that CP-2 did not appear to originate from myoid cells or dispersed germ cells but could be recovered from Sertoli cell-enriched cultures prepared from Stage VI tubules. Protein secretion by tubular segments was also characterized by immunoprecipitation with two polyspecific antisera directed against Sertoli cell products. Five secretory proteins were identified which had cycles different from one another and from CP-2. In contrast to secreted products, the synthesis of most cellular proteins by tubular segments remained relatively constant throughout the cycle. It is concluded: 1) segments of the seminiferous epithelium secrete proteins into the culture medium which are distinct from cellular proteins; 2) the synthesis of many of these proteins varies with the epithelial cycle; and 3) several of the secreted proteins are of Sertoli cell origin, including a newly identified protein, CP-2. This indicates that the morphology and the protein synthetic capacity of the seminiferous epithelium are coordinated over space and time.     

Subcellular and Perisynaptic Distribution of Rat Brain Aldehyde Dehydrogenase Activity

Abstract: The nuclear mitochondrial and synaptosomal fractions of rat brain were each found to contain some 25–30% of the total aldehyde dehydrogenase activity. The cytoplasmic fraction had a very low total aldehyde dehydrogenase activity. There were differences in the distribution of the activity when different aldehydes were used as substrates, suggesting the presence of isoenzymes in the various subcellular compartments. When rats were treated intra-cisternally with 6-hydroxydopamine there was no change in brain aldehyde dehydrogenase activity, although the noradrenaline content and the activities of tyrosine hydroxylase and dopamine-β-hydroxylase were markedly decreased. Treatment with 6-hydroxydopamine also had no significant effect on the aldehyde dehydrogenase activity in retinal homogenates. The results suggest that the aldehyde dehydrogenase activity in rat brain is predominantly outside the catecholaminergic nerve terminals.     

Comparison of the phenotypes of the lpxA and lpxD mutants of Escherichia coli

Abstract We compared the phenotype of two thermosensitive Escherichia coli mutants defective in lipid A biosynthesis i.e. SM101 ( lpxA ) and CDH23-213 ( lpxD ). More than 40% of the periplasmic 27-kDa marker enzyme β-lactamase was released from SM101 at 28°C. At this temperature, the mutant still grew with a generation time (67 min), not much longer than that of the parent control strain (57 min). CDH23-213 released β-lactamase only at higher temperatures. SM101 and CDH23-213 were both unable to grow in hypo-osmotic conditions. Derivatives of SM101 and CDH23-213 with mdoA ::Tn 10 had identical phenotypes (including thermosensitivity and defective outer membrane permeability barrier to hydrophobic probes) to those of SM101 and CDH23-213, indicating that the potential loss of membrane-derived oligosaccharides (MDO) did not explain these phenotypic properties. A method for the estimation of lipid A synthesis rate was developed.     

Recognition of balance signals between healthy subjects and otoneurological patients with hidden Markov models

The objective of the present research was to investigate whether hidden Markov models can be used to recognise and classify balance signals extracted from two subject groups, the healthy and patients suffering from otoneurological vertiginous diseases. Two different testing protocols were applied: arising from a chair and standing on the force platform. Signals recorded according to these protocols were trained for models with different numbers of states to find the best choices as model structures. We found that these models with 7–15 states were able to recognise the healthy subjects from the patients with the accuracy between 70 and 90% although their balance measurements were visually very similar and difficult to separate between two groups.     

Bacteria, phages and septicemia

The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.     

Inhibition of Aurora kinases perturbs chromosome alignment and spindle checkpoint signaling in rat spermatocytes

In somatic cells, integrity of cell division is safeguarded by the spindle checkpoint, a signaling cascade that delays the separation of sister chromatids in the presence of misaligned chromosomes. Aurora kinases play important roles in this process by promoting centrosome maturation, chromosome bi-orientation, spindle checkpoint signaling, and cytokinesis. To investigate the functions of Aurora kinases in male meiosis, we applied a small molecule Aurora inhibitor, ZM447439, to seminiferous tubules in vitro. Primary and secondary spermatocytes exposed to ZM447439 exhibit defects in the spindle morphology and fail to align their chromosomes at the metaphase plate. Moreover, the treated spermatocytes undergo a forced exit from the meiotic M-phase without cytokinesis. These results suggest that the activities of Aurora kinases are required for normal spindle assembly as well as for establishment and maintenance of proper microtubule-kinetochore attachments and spindle checkpoint signaling in male mammalian meiosis.     

AllerHunter: A SVM-Pairwise System for Assessment of Allergenicity and Allergic Cross-Reactivity in Proteins

Allergy is a major health problem in industrialized countries. The number of transgenic food crops is growing rapidly creating the need for allergenicity assessment before they are introduced into human food chain. While existing bioinformatic methods have achieved good accuracies for highly conserved sequences, the discrimination of allergens and non-allergens from allergen-like non-allergen sequences remains difficult. We describe AllerHunter, a web-based computational system for the assessment of potential allergenicity and allergic cross-reactivity in proteins. It combines an iterative pairwise sequence similarity encoding scheme with SVM as the discriminating engine. The pairwise vectorization framework allows the system to model essential features in allergens that are involved in cross-reactivity, but not limited to distinct sets of physicochemical properties. The system was rigorously trained and tested using 1,356 known allergen and 13,449 putative non-allergen sequences. Extensive testing was performed for validation of the prediction models. The system is effective for distinguishing allergens and non-allergens from allergen-like non-allergen sequences. Testing results showed that AllerHunter, with a sensitivity of 83.4% and specificity of 96.4% (accuracy = 95.3%, area under the receiver operating characteristic curve AROC = 0.928±0.004 and Matthew''s correlation coefficient MCC = 0.738), performs significantly better than a number of existing methods using an independent dataset of 1443 protein sequences. AllerHunter is available at http://tiger.dbs.nus.edu.sg/AllerHunter