Protein synthesis and secretion in human decidua of early pregnancy

Proteins synthesized and secreted by first trimester decidua in primary culture were identified. Explants were cultured for 24 h, in RPMI-1640 or Dulbecco's Modified Eagles Medium containing the radioactive amino acid 35S-methionine or 3H-proline. Electron microscopy of explants before and after 24 h of culture demonstrated the relative purity of the decidua, maintenance of cell integrity, and ultrastructural features indicative of active protein synthesis and secretion. Proteins synthesized and secreted by the explants into the medium were analyzed by fluorography of one-dimensional polyacrylamide gels in the presence of sodium dodecyl sulfate. By comparison of radiolabeled proteins from four women, eight 35S-methionine-labeled bands of 78, 70, 60, 50, 43, 34, 25, and 23 kDa were identified as common decidual peptides. A comparison of autoradiographs of the medium from decidual cultures to decidual cell homogenates showed that seven of these peptides were enriched in the culture medium. When labeled peptides from fibroblast cultures were compared to labeled proteins from decidual cultures each of the common decidual peptides (except the 70 kDa protein) occurred only in the decidual culture medium. Comparison of 3H-proline and 35S-methionine-labeled decidual proteins revealed that the 78, 70, 60, 50, and 34 kDa proteins were of similar fluorographic intensity when labeled with the two different amino acids. The 43, 25, and 23 kDa proteins appeared to contain more methionine, and proteins at 36, 20, 13, and 12 kDa were proline-rich, but contained less methionine. The seven decidual explant-specific, 35S-methionine-labeled secreted proteins were concentrated and purified by preparative gel electrophoresis, and antisera were generated to four of the putative decidual secretory proteins.     

DNS-Reduplikationsmuster der Somatischen Chromosomen von Cricetus cricetus (L.)

The patterns of terminal DNA synthesis of the autosomes and sex chromosomes of Cricetus cricetus were studied. Characteristic late replicating segments are found on all chromosomes allowing identification of most autosomes. The sex chromosomes of both sexes behave similarly; in the male, half of the X and the entire Y are late replicating and heteropycnotic, in the female, half of one X and the whole of the other X. The isopycnotic part of the X-chromosome comprises about 5% of the haploid female complement.

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Wesentliche Teile der vorliegenden Arbeit werden von Fräulein Dorothee Hepp als Dissertation der Medizinischen Fakultät der Universität Freiburg i. Br. vorgelegt.

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Wir danken Dr. Susumu Ohno für kristiche des Munuskriptes und Fräulein Elke Faisst für ihre bei den experimentellen Arbeiten.     

Melanoma formation in xiphophorus: A model system for the role of receptor tyrosine kinases in tumorigenesis

Cancer is one of the most frequent fatal human diseases. It is a genetic disease, and molecular analysis of the genes involved revealed that they belong to several distinct classes of molecules, one of which is the receptor tyrosine kinases. Neoplastic transformation is regarded as the result of a multistep process and, in most cases, it is hard to evaluate what the initial events in tumor formation are. What makes it difficult to approach this question is the paucity of animal models for tumorigenesis allowing investigation of the mechanisms leading to uncontrolled cell proliferation. Melanoma formation in Xiphophorus is one of these model systems. Here, overexpression and activation of a receptor tyrosine kinase causes neoplastic transformation of pigment cells. Xiphophorus provides all the advantages of a well-characterised genetic system. In addition, some crucial components of the transformation pathway have been identified at the molecular level. As a vertebrate, Xiphophorus might serve as a model system to aid understanding, in more general terms, of the mechanisms of tumorigenesis in human diseases.     

The hypoosmotic swelling test performed with coulter counter: a method to assay functional integrity of sperm membrane in rainbow trout.

The hypoosmotic swelling test (HOS) is one of the methods used to evaluate sperm quality in mammals. This test is based on the swelling ability that functional spermatozoa have when submitted to hypoosmotic solutions. Only a slight increase in size is caused in rainbow trout spermatozoa in such conditions and it is not possible to distinguish between reactive cells (cells who were capable to increase in volume) and non-reactive cells (did not increase in volume) under light microscopy. In our approach we have used the coulter counter to verify the effectiveness of the HOS test in this species. Semen was diluted in different hypoosmotic solutions (50, 100, 150, 200, 250 and 320 mosM/kg) and cell volume measured at different times after dilution (30 s, 2, 5, 10, 20, and 30 min). The higher percentage of reactive cells was achieved with the 100 mosM/kg solution and swelling occurred before 30 s. Even with this solution, the small increase in cell size caused the overlapping of volumes from swollen and non-swollen spermatozoa. In order to analyse the data and to choose a parameter suitable for assessing cell reactivity, the test was performed in samples containing known rates of live/dead cells. Two parameters were analysed after swelling: the increase in volume and the percentage of cells over a standard volume (reactive cells). Results showed a high correlation between the percentages of reactive cells and the known rate of live cells (r2 = 0.65). This fact suggests that HOS test could be used to analyse the integrity and functionality of rainbow trout fresh sperm. To study the reliability of this test in cryopreserved sperm, simple linear regressions were made between cell viability determined by Hoechst 33285 dye and the two parameters obtained from coulter counter data. No significant correlation was observed in either case, showing that structural and functional integrity do not correlate after freeze/thaw. Consistently, the HOS test is not a reliable method to evaluate cryopreserved sperm quality in rainbow trout.     

Structure of viral φ29 DNA condensed by simple triamines: A light-scattering and electron-microscopy study

The structures of viral φ29 DNA condensed by triamines, principally spermidine, in 10^?3M NaCl were investigated by static and dynamic light scattering and electron microscopy. All of the results for DNA condensed in 30 μM spermidine at neutral pH are quantitatively consistent with a toroidal structure with a mean outer diameter of 1850 Å. At pH 10.2, however, condensed structures of a completely different size and shape are observed for the first time. These structures are also more irregular in shape and more polydisperse than those at neutral pH. This conformational change is believed to result from a change in the mode of spermidine binding that is coupled to, or associated with, the (premature) titration of protons on the base-ring nitrogens of guanine and thymine. Besides spermidine, certain homologs of spermidine, in which the butyl moiety of spermidine was replaced by longer pentyl through octyl moieties, were also studied. Though all of the triamines condensed the DNA at 30 μM, aggregation became a more prevalent occurrence as the length of the end chain increased. This suggests that crosslinking may play an important role in the condensation process. Finally, these aggregates are dissociated to a considerable extent at pH 10.2, and the resulting compact structures appear to be quite similar, independent of the triamine used to condense the DNA. The observed partial breakdown of aggregates is also consistent with the hypothesis of a change in mode of triamine binding at pH 10.2.     

Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> molecular epidemiology studies

Background  

During the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR (Mycobacterial interspersed repetitive units- variable number of tandem repeats) (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format.     

Use of the Four-and-a-Half Clearing Technique to Study Gymnosperm EmbryoIogy: Cunninghamia lanceolata

Since its introduction in 1971, the four-and-a-half clearing technique has been widely applied to the study of ovule and female gametophyte development in flowering plants as an alternative to the more arduous paraffin section methods. The technique has undergone several modifications that have broadened its application in studies of Angiosperm embryology. To date, however, the technique has not been successfully applied to embryological features of Gymnosperms. Dark coloration caused by naturally occurring substances and by-products of fixation render the clearing fluid ineffective, and special pretreatment methods used to remove dark substances in Angiosperm ovules have little or no effect on Gymnosperm material. In the technique reported here, paraffin sections of ovules and young seeds of Cunninghamia lanceolata 80-120 μm thick are cleared in benzyl benzoate-412 clearing fluid and examined with phase contrast optics. Observations of the mature female gametophyte in these cleared preparations are compared with those obtained from 10 μm sections, stained with safranin and fast green, and examined with bright-field optics. Although contrast and definition are more pronounced in stained sections than in cleared ones, the differences would not alter one's interpretation of characteristic structural features. The thick, cleared section offers an advantage over the thin, stained one in that many structural entities are contained within a single section rather than spread through several serial sections. The time required for clearing thick sections is much shorter than that required for making permanent stained preparations.