Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: mechanisms of haploid gene product sharing

Stable cytoplasmic bridges (or ring canals) connecting the clone of spermatids are assumed to facilitate the sharing of haploid gene products and synchronous development of the cells. We have visualized these cytoplasmic bridges under phase-contrast optics and recorded the sharing of cytoplasmic material between the spermatids by a digital time-lapse imaging system ex vivo. A multitude of small (ca. 0.5 microm) granules were seen to move continuously over the bridges, but only 28% of those entering the bridge were actually transported into other cell. The average speed of the granules decreased significantly during the passage. Immunocytochemistry revealed that some of the shared granules contained haploid cell-specific gene product TRA54. We also demonstrate the novel function for the Golgi complex in acrosome system formation by showing that TRA54 is processed in Golgi complex and is transported into acrosome system of neighboring spermatid. In addition, we propose an intercellular transport function for the male germ cell-specific organelle chromatoid body. This mRNA containing organelle, ca. 1.8 microm in diameter, was demonstrated to go over the cytoplasmic bridge from one spermatid to another. Microtubule inhibitors prevented all organelle movements through the bridges and caused a disintegration of the chromatoid body. This is the first direct demonstration of an organelle traffic through cytoplasmic bridges in mammalian spermatogenesis. Golgi-derived haploid gene products are shared between spermatids, and an active involvement of the chromatoid body in intercellular material transport between round spermatids is proposed.     

Increased substitution of phosphate groups in lipopolysaccharides and lipid A of the polymyxin-resistant pmrA mutants of Salmonella typhimurium: a 31P-NMR study

De-O-acylated lipopolysaccharides (LPS) of three polymyxin-resistant Salmonella typhimurium pmrA mutants and their parent strains were analysed by ³¹P-NMR (nuclear magnetic resonance) in order to assess, in relation to polymyxin resistance, the types and degree of substitution of phosphates of the LPS and lipid A. in the pmrA mutant LPS phosphate diesters predominated over phosphate monoesters, whereas the latter were more abundant in the parent wild-type LPS. The increase in the proportion of phosphate diesters was traced to both the core oligosaccharide and the lipld A part. In the latter, the ester-linked phosphate at position 4’was to a large extent (79–88%) substituted with 4-amino-4-deoxy-l -arabinose, whereas in the wild-type LPS the 4′-phosphate was mainly present as monoester. In each LPS, regardless of the pmrA mutation, the glycosidically linked phosphate of lipid A was largely unsubstituted.     

Soft-tissue sarcomas of the upper extremity: surgical treatment and outcome

The objective of this retrospective follow-up study was to evaluate the outcome of patients with soft-tissue sarcoma treated by the authors' protocol, which consists of a selective combination of conservative surgery and radiotherapy. Patients who relapsed were especially evaluated to improve treatment results. The authors examined 80 patients with local soft-tissue sarcoma in the upper extremity referred to their multidisciplinary group. Fifteen patients were referred for first or subsequent local recurrence, and 65 patients were treated for primary tumor. The goal of treatment was local control and preservation of a functional limb. Wide excision was attempted. If the margin was less than 2.5 cm, postoperative radiotherapy was administered. Eighty-five percent of the patients were treated by limb salvage. Thirty patients needed reconstructive procedures such as pedicled (20 patients) or free flaps (10 patients). No free flaps were lost. The 5-year disease-specific overall survival rate was 75 percent, the local recurrence-free survival rate was 79 percent, and the metastasis-free survival rate was 68 percent. In univariate analysis, prognostic factors for local recurrence were extracompartmental site; for development of metastases, large size and extracompartmental site; and for decreased disease-specific overall survival, large size and extracompartmental site. Intramuscular, cutaneous, and subcutaneous tumors had a 5-year local control rate of 100 percent, and extracompartmental tumors had a local control rate of 69 percent. Extracompartmental tumors clearly have the worst prognosis and should be the main target for improving treatment strategies. After exclusion of patients with inadequate treatment according to the authors' protocol, the local control rate at 5 years was 90 percent. Strict adherence to treatment protocol should be practiced.     

Decomposition of cellulose strips in relation to climate,litterfall nitrogen,phosphorus and C/N ratio in natural boreal forests

Isolates of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, Penicillium sp., Rhizoctonia solani, Stemphylium sp., Thielaviopsis basicola, and Verticillium dahliae were cultured on potato–dextrose agar (PDA), barley-sand and alfalfa-sand substrates in petri-dish or in column microcosms. N-mineralization by fungi and fungal-feeding nematodes in combination or fungi alone was assessed. Numbers of Aphelenchus avenae or Aphelenchoides composticola supported by the fungi were measured every 7 days. Times for full colonization of the substrates by fungi ranged from 5 to 15 days. Rhizoctonia solani and B. cinerea on PDA supported the largest A. avenae and A. composticola populations, respectively. Penicillium sp. was a nonhost for A. composticola and A. avenae. Rhizoctonia solani, B. cinerea, V. dahliae, and F. oxysporum supported significantly more nematodes than the other four fungal species. The ranked order of fungi based on the amount of N mineralized in columns free of nematodes was A. alternata (with a rate of 0.052 μg N/g-sand per day), Stemphylium sp., V. dahliae, T. basicola, B. cinerea, F. oxysporum, R. solani, and Penicillium sp. (with a rate of 0.0045 μg N/g-sand perday). The presence of A. avenae resulted in significant increases in mineral N, compared to nematode-free columns colonized by F. oxysporum, R. solani, and T. basicola alone. The presence of A. composticola resulted in significant increases in mineral N, compared to nematode-free columns colonized by A. alternata, B. cinerea, F. oxysporum, and R. solani alone. There was more mineral N incolumns in the presence of A. composticola than A. avenae in most cases. This revised version was published online in August 2006 with corrections to the Cover Date.     

Novel Analysis Software for Detecting and Classifying Ca2+ Transient Abnormalities in Stem Cell-Derived Cardiomyocytes

Comprehensive functioning of Ca²⁺ cycling is crucial for excitation–contraction coupling of cardiomyocytes (CMs). Abnormal Ca²⁺ cycling is linked to arrhythmogenesis, which is associated with cardiac disorders and heart failure. Accordingly, we have generated spontaneously beating CMs from induced pluripotent stem cells (iPSC) derived from patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), which is an inherited and severe cardiac disease. Ca²⁺ cycling studies have revealed substantial abnormalities in these CMs. Ca²⁺ transient analysis performed manually lacks accepted analysis criteria, and has both low throughput and high variability. To overcome these issues, we have developed a software tool, AnomalyExplorer based on interactive visualization, to assist in the classification of Ca²⁺ transient patterns detected in CMs. Here, we demonstrate the usability and capability of the software, and we also compare the analysis efficiency to manual analysis. We show that AnomalyExplorer is suitable for detecting normal and abnormal Ca²⁺ transients; furthermore, this method provides more defined and consistent information regarding the Ca²⁺ abnormality patterns and cell line specific differences when compared to manual analysis. This tool will facilitate and speed up the analysis of CM Ca²⁺ transients, making it both more accurate and user-independent. AnomalyExplorer can be exploited in Ca²⁺ cycling analysis to study basic disease pathology and the effects of different drugs.     

The effect of oligolysines Lys-3, Lys-4, and Lys-5 on the outer membrane permeability of Pseudomonas aeruginosa

A lysine polymer with five residues (Lys-5) was found to remarkably increase the outer membrane (OM) permeability of Pseudomonas aeruginosa to the tested hydrophobic probes (nitrocefin, N-phenyl naphthylamine, rifampin). Lys-3 and Lys-4 were inactive. The OM of Escherichia coli and Salmonella typhimurium was not permeabilized by Lys-5. Furthermore, even the action of Lys-5 on the Pseudomonas OM was abolished when the assays were performed in the presence of 150 mM NaCl instead of the low-ionic strength buffer earlier used by investigators studying the effect of polycations on the Pseudomonas OM.     

Fish diversity in European lakes: geographical factors dominate over anthropogenic pressures

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Characterization,bioinformatic analysis and dithiocarbamate inhibition studies of two new α-carbonic anhydrases,CAH1 and CAH2, from the fruit fly Drosophila melanogaster

Carbonic anhydrases (CAs) are essential and ubiquitous enzymes. Thus far, there are no articles on characterization of Drosophila melanogaster α-CAs. Data from invertebrate CA studies may provide opportunities for anti-parasitic drug development because α-CAs are found in many parasite or parasite vector invertebrates. We have expressed and purified D. melanogaster CAH1 and CAH2 as proteins of molecular weights 30 kDa and 28 kDa. CAH1 is cytoplasmic whereas CAH2 is a membrane-attached protein. Both are highly active enzymes for the CO₂ hydration reaction, being efficiently inhibited by acetazolamide. CAH2 in the eye of D. melanogaster may provide a new animal model for CA-related eye diseases. A series of dithiocarbamates were also screened as inhibitors of these enzymes, with some representatives showing inhibition in the low nanomolar range.     

Analysis of evolution of carbonic anhydrases IV and XV reveals a rich history of gene duplications and a new group of isozymes

Carbonic anhydrase (CA) isozymes CA IV and CA XV are anchored on the extracellular cell surface via glycosylphosphatidylinositol (GPI) linkage. Analysis of evolution of these isozymes in vertebrates reveals an additional group of GPI-linked CAs, CA XVII, which has been lost in mammals. Our work resolves nomenclature issues in GPI-linked fish CAs. Review of expression data brings forth previously unreported tissue and cancer types in which human CA IV is expressed. Analysis of collective glycosylation patterns of GPI-linked CAs suggests functionally important regions on the protein surface.