Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2^−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2^−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2^−/−mammary fat pad showed a decrease in the content of myristic acid (C₁₄), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2^−/− mice.     

Strong antimutagenic effects of fluoride on mutation induction by trenimon and 1-phenyl-3,3-dimethyltriazene in Drosophila melanogaster

After fluoride treatment of mature and immature oocytes of Drosophila females, a clear-cut dose-dependent decrease in fertility and fecundity was observed. The hatchability of mature oocytes was reduced by as much as 35%. When immature oocytes were treated, a pronounced dose-dependent reduction in fecundity occurred.

Exposure of mature sperm to NaF resulted in a slight decrease in fertility, comparable to the effect obtained with immature oocytes. Of the criteria used to measure possible mutagenic effects of NaF (sex-linked lethals, partial and total X- and Y-chromosome losses), only the rate of total losses was enhanced significantly.

The slight mutagenic effect of NaF on mature sperm was not related to the strong antimutagenic activity observed, when applied simultaneously with any of the several chemical mutagens. NaF treatment drastically reduced both the Trenimon-induced decrease in fertility and Trenimon-induced increases in recessive lethal mutation frequencies and rates of partial and total chromosome losses. The inhibitory effect of NaF was less pronounced with 1-phenyl-3,3-dimethyltriazene (PDT), a poor chromosome breaker in Drosophila, and absent for A 137, a weak mutagen which so far has failed to induce chromosomal aberrations in Drosophila. Therefore, the data are interpreted as being the result of a specific fluoride inhibition of chemically induced chromosomal breakage.

In mature and immature oocytes, the decreases in fertility and fecundity, and increase in recessive lethal frequency (mature oocytes) produced by Trenimon were also suppressed in the presence of fluoride. However, since Trenimon failed to produce a significant rise in X losses and NDJ in both stages, the effect of NaF on these mutational classes was, of course, not testable.     

A vector for recombinant DNA in Staphylococcus aureus

Staphylococcal plasmids pS194 and pSC194 which confer streptomycin and streptomycin-chloramphenicol resistance respectively have been used as vectors for construction of recombinant DNA, since they each carry one single recipient site for endonuclease EcoRI. Hybrid DNA does not express streptomycin resistance, a marker which is present in both vectors, presumably because the marker gene is cleaved by EcoRI. A chloramphenicol marker present in pSC194 was used for positive hybrid selection. Hybrid plasmids generated by joining pSC194 with one or more of the four EcoRI fragments of the large (18.1-10(6) daltons) staphylococcal plasmid pI258 were constructed and permitted us to develop a physical map for pI258.     

Transcription termination: sequence and function of the rho-independent tL3 terminator in the major leftward operon of bacteriophage lambda

For cloning, assaying the function and sequencing terminators, we have constructed the pD12 plasmid, in which the late promotor p'R of phage lambda controls the expression of the galK gene of the pK03 plasmid of McKenney et al. (1981). The lambda tL3 terminator region was cloned in this plasmid between the promoter and the galK gene, and found to be 90-94% effective in preventing galactokinase expression in both rho+ and rho- hosts. Is is also active in vitro, both in the presence or absence of the rho factor. The termination point is located at 4320 bp to the left of the SL startpoint of the PL-RNA, just downstream of gene exo. We have sequenced 356 bp of the hitherto uncharted lambda DNA to the right of the TaqI cut, which in turn is 110 bp to the right of the b522 deletion at 63.9% lambda. The tL3 terminator has several features common to other rho-independent termination sequences, including an 81% G+C-rich region of 2X8-bp symmetry ("stem") with a 5-bp intervening "loop", partially overlapping and followed by a sequence transcribed into the pyrimidine-rich CCUUUCU-OH 3' terminus of the RNA. The termination point that follows the last U was determined by the S1 mapping technique.     

An unusual transmembrane helix in the endoplasmic reticulum ubiquitin ligase Doa10 modulates degradation of its cognate E2 enzyme

In the endoplasmic reticulum (ER), nascent membrane and secreted proteins that are misfolded are retrotranslocated into the cytosol and degraded by the proteasome. For most ER-associated degradation (ERAD) substrates, ubiquitylation is essential for both their retrotranslocation and degradation. Yeast Doa10 is a polytopic membrane ubiquitin ligase (E3) that along with its cognate ubiquitin-conjugating enzymes (E2s), Ubc7 and the C-terminally membrane-anchored Ubc6, makes a major contribution to ER-associated degradation. Ubc6 is also a substrate of Doa10. One highly conserved Doa10 element, the uncharacterized ~130-residue TEB4-Doa10 domain, includes three transmembrane helices (TMs). We find that the first of these, TM5, includes an absolutely conserved ΦPΦXXG motif that is required for Doa10 function, as well as highly conserved negatively charged glutamate and aspartate residues. The conservative exchange of the TM5 glutamate to aspartate (doa10-E633D) results in complete stabilization of Ubc6 but has little if any effect on other substrates. Unexpectedly, mutating the glutamate to glutamine (doa10-E633Q) specifically accelerates Ubc6 degradation by ~5-fold. Other substrates are weakly stabilized in doa10-E633Q cells, consistent with reduced Ubc6 levels. Notably, catalytically inactive ubc6-C87A is degraded in doa10-E633Q but not wild-type cells, but an active version of Ubc6 is required in trans. Fusion of the Ubc6 TM to a soluble protein yields a protein that is degraded in a doa10-E633Q-dependent manner, whereas fusion of the C-terminal TM from an unrelated protein does not. These results suggest that the TEB4-Doa10 domain regulates Doa10 association with the Ubc6 membrane anchor, thereby controlling the degradation rate of the E2.     

PI3K signaling in the regulation of branching morphogenesis

Branching morphogenesis drives the formation of epithelial organs including the mammary gland, lung, kidney, salivary gland and prostate. Branching at the cellular level also drives development of the nervous and vascular systems. A variety of signaling pathways are orchestrated together to establish the pattern of these branched organs. The phosphoinositide 3-kinase (PI3K) signaling network is of particular interest because of the diverse outcomes it generates, including proliferation, motility, growth, survival and cell death. Here, we focus on the role of the PI3K pathway in the development of branched tissues. Cultured cells, explants and transgenic mice have revealed that the PI3K pathway is critical for the regulation of cell proliferation, apoptosis and motility during branching of tissues.     

Fluoride: interaction with chemical mutagens in Drosophila

Simultaneous feeding of sodium fluoride and some chemical mutagens to Drosophila has been reported to reduce the yield of induced mutations compared with feeding the mutagens alone. This observation has been interpreted as a genuine case of antimutagenesis in which fluoride specifically inhibits the induction of chromosome breaks. An alternative hypothesis is that the presence of fluoride inhibits the uptake of the mutagen solutions, producing the same effect as an antimutagen, but for a trivial reason. We have tested this hypothesis using radioactive labelled sucrose to measure the uptake of test solutions. The results show that differential uptake can account for the "antimutagenic" effects reported in Drosophila. Comparison of recessive lethal frequencies induced by Trenimon and PDT do not support the hypothesis that fluoride has any genuine antimutagenic action. Antimutagenic effects of fluoride have been reported in other systems. We cannot exclude the possibility of some genuine effects but we consider that these reports should be critically re-examined in the light of our present findings.     

CYP3A4 ubiquitination by gp78 (the tumor autocrine motility factor receptor, AMFR) and CHIP E3 ligases

Human liver CYP3A4 is an endoplasmic reticulum (ER)-anchored hemoprotein responsible for the metabolism of >50% of clinically prescribed drugs. After heterologous expression in Saccharomyces cerevisiae, it is degraded via the ubiquitin (Ub)-dependent 26S proteasomal pathway that utilizes Ubc7p/Cue1p, but none of the canonical Ub-ligases (E3s) Hrd1p/Hrd3p, Doa10p, and Rsp5p involved in ER-associated degradation (ERAD). To identify an Ub-ligase capable of ubiquitinating CYP3A4, we examined various in vitro reconstituted mammalian E3 systems, using purified and functionally characterized recombinant components. Of these, the cytosolic domain of the ER-protein gp78, also known as the tumor autocrine motility factor receptor (AMFR), an UBC7-dependent polytopic RING-finger E3, effectively ubiquitinated CYP3A4 in vitro, as did the UbcH5a-dependent cytosolic E3 CHIP. CYP3A4 immunoprecipitation coupled with anti-Ub immunoblotting analyses confirmed its ubiquitination in these reconstituted systems. Thus, both UBC7/gp78 and UbcH5a/CHIP may be involved in CYP3A4 ERAD, although their relative physiological contribution remains to be established.     

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus with high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site And two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).