Indirect Sertoli cell-mediated ablation of germ cells in mice expressing the inhibin-alpha promoter/herpes simplex virus thymidine kinase transgene

In the present study, we describe a novel mouse model for inducible germ cell ablation. The mice express herpes simplex virus thymidine kinase (HSV-TK) under the inhibin-alpha subunit promoter (Inhalpha). When adult transgenic (TG) mice were treated with famciclovir (FCV) for 4 wk, their spermatogenesis was totally abolished, with only Sertoli cells and few spermatids remaining in the seminiferous tubules. However, testicular steroidogenesis was not affected. Shorter treatment periods allowed us to follow up the progression of germ cell death: After 3 days, spermatogonia and preleptotene spermatocytes were no longer present. After a 1-wk treatment, spermatogonia, preleptotene, and zygotene spermatocytes were missing and the amount of pachytene spermatocytes was decreased. After a 2-wk treatment, round and elongating spermatids were present. During the third week, round spermatids were lost and, finally, after a 4-wk treatment, only Sertoli cells and few spermatids were present. Interestingly, the transgene is detected in Leydig and Sertoli cells but not in spermatogonia. This suggests that FCV is phosphorylated in Sertoli cells, and thereafter, leaks to neighboring spermatogonia, apparently through cell-cell junctions present, enabling trafficking of phosphorylated FCV. Because of the many mitotic divisions they pass through, the spermatogonia are very sensitive to toxins interfering with DNA replication, while nondividing Sertoli cells are protected. Using transillumination-assisted microdissection of the seminiferous tubules, the gene-expression patterns analyzed corresponded closely to the histologically observed progression of cell death. Thus, the model offers a new tool for studies on germ cell-Sertoli cell interactions by accurate alteration of the germ cell composition in seminiferous tubules.     

The Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane as an Artificial Nucleobase

{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4′-dimethoxytrityl)-α-D- erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1- yl]ethyl}acetamide (1) was prepared and incorporated into a 2′-O-methyl oligoribonucleotide. The hybridization of this oligonucleotide with complementary 2′-O-methyl oligoribonucleotides incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence and presence of Zn²⁺. Introduction of Zn²⁺ moderately stabilized the duplex with U-bulged targets.     

Spring development of a Chlamydomonas population in Lake Nimetön,a small humic forest lake in southern Finland

Biomass development and vertical distribution of a Chlamydomonas population in a small humic forest lake was followed by daily sampling in May-June, 1984. Chlamydomonas dominated the phytoplankton spring bloom, forming 71% of the maximum phytoplankton biomass on 18 May. In early May the outflow rate was high and during the 24 hour period when the maximum rate of surface runoff was recorded (8–9 May), 43% of the Chlamydomonas biomass was flushed out of the lake, which delayed the onset of biomass increase. When surface runoff had slowed down Chlamydomonas biomass started increasing and during wax of the population most cells were < 10 µm in diameter. Population maximum lasted for one day (18 May) and there-after Chlamydomonas biomass decreased towards the end of the study. During wane of the population most cells were > 10 µm in diameter.     

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.     

Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.