Cell death by apoptosis during involution of the lactating breast in mice and rats

The role of cell death in involution of lactating breast was investigated in mice and rats by light and electron microscopy. Apoptosis, recognized by sharply demarcated compaction of chromatin against the nuclear envelope and by shrinkage and budding of the whole cell to form membrane-bounded apoptotic bodies, was responsible for major loss of cells in both species. In the mouse, rapid involution during the first 2 days was associated with shedding of large numbers of apoptotic bodies derived from alveolar epithelial cells into alveolar lumens. This was followed by more gradual regression, during which the bodies were mostly phagocytosed by macrophages within the epithelium. In the rat, glandular involution was a more gradual and uniform process, with shedding of apoptotic epithelial cells into alveolar lumens being much less conspicuous. Apoptosis of myoepithelial cells was observed in mice, the resulting apoptotic bodies being phagocytosed by intraepithelial macrophages, but was not detected in rats. Apoptosis of capillary endothelial cells caused rapid regression of the capillary beds in both mice and rats. Intraepithelial macrophages increased in number during involution, developed cytoplasmic lipofuscin pigment, and either remained within the epithelium or migrated to the interstitium and regional nodes. Cell loss by apoptosis has been demonstrated during involution and atrophy of a variety of other glands. It characteristically results in shrinkage of a tissue without disruption of its basic architecture.     

Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes

Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.     

The spectrin-actin junction of erythrocyte membrane skeletons

High-resolution electron microscopy of erythrocyte membrane skeletons has provided striking images of a regular lattice-like organization with five or six spectrin molecules attached to short actin filaments to form a sheet of five- and six-sided polygons. Visualization of the membrane skeletons has focused attention on the (spectrin)5,6-actin oligomers, which form the vertices of the polygons, as basic structural units of the lattice. Membrane skeletons and isolated junctional complexes contain four proteins that are stable components of this structure in the following ratios: 1 mol of spectrin dimer, 2-3 mol of actin, 1 mol of protein 4.1 and 0.1-0.5 mol of protein 4.9 (numbers refer to mobility on SDS gels). Additional proteins have been identified that are candidates to interact with the junction, based on in vitro assays, although they have not yet been localized to this structure and include: tropomyosin, tropomyosin-binding protein and adducin. The spectrin-actin complex with its associated proteins has a key structural role in mediating cross-linking of spectrin into the network of the membrane skeleton, and is a potential site for regulation of membrane properties. The purpose of this article is to review properties of known and potential constituent proteins of the spectrin-actin junction, regulation of their interactions, the role of junction proteins in erythrocyte membrane dysfunction, and to consider aspects of assembly of the junctions.     

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.     

Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.     

Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase.

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.     

Translocation ofLactobacillus murinus from the gastrointestinal tract

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other extraintestinal sites. The translocation rate of a newly described species of indigenous bacteria,Lactobacillus murinus, was compared with the translocation rates of indigenousLactobacillus acidophilus and nonindigenousSalmonella enteritidis. Groups of germfree or antibiotic-decontaminated, specific pathogen-free mice were monoassociated with each of these bacterial strains and tested at various intervals for translocation to the mesenteric lymph nodes. The translocation rates of the various bacteria expressed in decreasing order as the numbers of translocating bacteria per gram mesenteric lymph node wereS. enteritidis, L. murinus, andL. acidophilus. The degree of histologic damage to the gastrointestinal mucosa after monoassociation with these strains followed the same pattern. Thus,L. murinus translocates from the GI tract at a surprisingly high rate for an indigenous bacterial strain, and its translocation appears to be associated with mucosal alterations.     

Kinetics and location of bone marrow cell graft rejection by irradiated mice

Natural killer (NK) cells were eliminated with rabbit anti-Asialo GM1 (anti-ASGM1) serum to test the kinetics and location of bone marrow cell (BMC) rejection. Anti-ASGM1 serum was injected intravenously in mice at various times before or after irradiation (8.6 Gy) and transfer of parental-strain or allogeneic BMC. Growth of BMC was determined by measuring splenic 5-iodo-2'-deoxyuridine-125I incorporation 5 days after cell transfer. Anti-ASGM1 serum weakened hybrid resistance even if injected intravenously as late as 24 h post-BMC transfer and even in recipients injected with polyinosinic:polycytidylic acid so as to boost NK activity. If regenerating spleen cells (higher rate of cell cycling) were used as donor cells instead of BMC, the length of time required for rejection was unaffected. Anti-ASGM1 serum injected intravenously rapidly inhibited splenic NK activity and lung clearance of YAC-1 tumor cells, but when injected intratracheally, it only inhibited lung NK activity. Thus, BMC rejection occurs in the hematopoietic tissue and requires at least 24 h.     

Release of putative exocytic transport vesicles from perforated MDCK cells.

Mechanically perforated MDCK cells were used to study membrane transport between the trans-Golgi network and the apical and basolateral plasma membrane domains in vitro. Three membrane transport markers--an apical protein (fowl plague virus haemagglutinin), a basolateral protein (vesicular stomatitis virus G protein), and a lipid marker destined for both domains (C6-NBD-sphingomyelin)--were each accumulated in the trans-Golgi by a 20 degrees C block of transport and their behaviour monitored following cell perforation and incubation at 37 degrees C. In the presence of ATP and in the absence of calcium ions a considerable fraction of the transport markers were released from the perforated cells in sealed membrane vesicles. Control experiments showed that the vesicles were not generated by non-specific vesiculation of the Golgi complex or the plasma membrane. The vesicles had well defined sedimentation properties and the orientation expected of transport vesicles derived from the trans-Golgi network.