Characterization of epithelial cell islets in primary monolayer cultures of human breast carcinomas by the tetrazolium reaction for glucose 6-phosphate dehydrogenase

Epithelial cell islets in primary monolayer cultures of human breast biopsies were characterized by combined immuno-, enzyme- and DNA cytochemistry as well as by analysis of attachment-, spread- and growth patterns. For cultivation we used explants from reduction mammoplasties, benign lesions, primary carcinomas and metastases. Milk fat globule membrane antigen (MFGM-A) was detected with a monoclonal antibody, and the tetrazolium reaction for glucose 6-phosphate dehydrogenase (G6PDH) as well as DNA content of the cultured cells were quantified. Spreading and growth of individual islets were studied by image analysis. Fibroblast-like cells did not express MFGM-A, and whereas epithelial (MFGM-A positive) cell islets of normal and benign origin showed cells with no or low G6PDH reaction, respectively, the majority of epithelial cell islets from 11 out of 21 carcinomas showed strong reaction. Cell islets with strong G6PDH reaction were sometimes hyperdiploid. Moreover, whereas cell islets with no or low reaction from both benign lesions and carcinomas readily attached and spread in a serum-free medium and showed population doubling times of 30 to 110 h, cell islets with strong reaction from carcinomas and metastatic lesions required serum for attachment and their growth rate was too low to be determined.     

Immunohistochemical distribution of MAM-3 and MAM-6 antigens in developing salivary glands of the human fetus

Summary The immunohistochemical expression of MAM-3 and MAM-6 antigens was studied in developing human fetal salivary gland removed at autopsy of 22 normal fetuses of varying maturity (10–40 weeks of gestation). The onset of functional maturation in the fetal gland was seen at 21 weeks of gestational maturity. The acini and ducts then underwent distinct alterations in antigen expression with growth and maturation until the late developmental stage (33–40 weeks of gestation) when they resemble the adult salivary gland. The role of maturing duct cells in histogenesis of salivary gland tumours is discussed.     

Gramicidin A induced fusion of large unilamellar dioleoylphosphatidylcholine vesicles and its relation to the induction of type II nonbilayer structures.

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.     

Sorbitol dehydrogenase genetics in the mouse: A ';null' mutant in a ';European' C57BL strain

A ';null' activity variant phenotype for sorbitol dehydrogenase (SDH) was observed in C57BL/LiA mice and used to examine the genetics of this enzyme. Linkage studies of the locus ( Sdh-1 ) with non-agouti (a) and a biochemical Iocus encoding liver L-α-hydroxyacid oxidase ( Hao-1 ) demonstrated that it is coincident with or closely linked to the structural locus, previously localized on chromosome 2. Alcohol dehydrogenase (ADH) isozymes were also examined, since the liver A₂ isozyme exhibited some activity as a sorbitol dehydrogenase on cellulose acetate zymograms. It is apparent that SDH activity is not ';essential' in this mouse strain.     

Identification of a mouse gene required for binding of Rauscher MuLV envelope gp70.

Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemai cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Ruascher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine luekemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1 (replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus.     

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.     

Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles of the motif for the voltage-dependent and voltage-independent gating, we performed reciprocal replacements of the asparagine and aspartate within the S3 motif in both channels, following the proposed idea that specific electrostatic interactions with other domains take place during gating. Wild-type and mutant channels were heterologeously expressed in HEK-293 cells and channel function was analyzed by whole-cell patch-clamp analysis as well as by Ca²⁺-imaging. Additionally, the expression of the channels in the plasma membrane was tested by Western blot analysis, in part after biotinylation. For the mutations of TRPM8, responses to menthol were only compromised if also the expression of the glycosylated channel isoform was prevented. In contrast, responses to cold were consistently and significantly attenuated but not completely abolished. For TRPM2, surface expression was not significantly affected by any of the mutations but channel function was only retained in one variant. Remarkably, this was the variant of which the corresponding mutation in TRPM8 exerted the most negative effects both on channel function and expression. Furthermore, we performed an exchange of the inner pair of residues of the N-x-x-D motif between the two channels, which proved deleterious for the functional expression of TRPM8 but ineffective on TRPM2. In conclusion, the N-x-x-D motif plays specific roles in TRPM8 and TRPM2, reflecting different requirements for voltage-dependent and voltage-independent channel gating.     

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation.     

Incomplete proviral genome of mouse mammary tumour virus is present on chromosome Y of NIH Swiss and some genetically related mouse strains.

An incomplete proviral genome of endogenous mammary tumour virus (MMTV) was found in DNA of several strains of mice. This MMTV-related sequence was assigned to the Y chromosome since it was clearly observed in male mice only. This MMTV provirus contained a sequence related to LTR (long terminal repeat), but not to gag-pol and env genes. NFS, NIH Swiss/S, STS/A, and DD/Tbr mice have this sequence but BALB/cHeA, SHN, SLN, C57BL/6NJcl, C3H/HeNJcl and CBA/JJcl mice are negative. In the strains containing this sequence, a DNA test for the sequence makes it possible to easily distinguish the DNAs of male or female mice.