Export and secretion of overproduced OmpA-beta-lactamase in Escherichia coli

The export of beta-lactamase to the periplasm of Escherichia coli can be directed by the OmpA signal peptide in the secretion cloning vector pIN-III. The overproduction of the hybrid precursor specifically induces a delay in the onset of processing of newly synthesized polypeptide chains. However, when the processing starts, no alteration in the rate of cleavage itself is observed. Our results suggest that the temporal mode of processing (which reflects translocation) does not depend on the nature of the signal peptide but rather depends on the nature of the polypeptide chain exported.     

A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli

Inducible hybrid genes encoding two large domains, a periplasmic domain consisting of the PhoS sequence and an outer membrane domain corresponding to various lengths of the OmpF mature sequence were constructed. The synthesized hybrid polypeptides are correctly processed during the early times of induction, their precursor forms being accumulated at later times. These hybrids restore sensitivity toward colicin A to ompF E coli B strain which suggests an outer membrane location. At least 2 of them are indeed localized in the outer membrane after immunogold labelling on ultrathin cryosections. Insertion of a hydrophobic sequence between PhoS and OmpF improves the trimerization and the assembly of the OmpF part. Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimeric conformation which is recognized by specific antibodies. Moreover, the deletion of the C-terminal region impairs the functional insertion of the OmpF domain; this indicates that the last membrane-spanning region of OmpF is necessary for the correct folding and orientation of the protein in the outer membrane.     

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.     

Age-associated changes in initiation of ribonucleic acid synthesis in isolated rat liver nuclei.

Initiation of RNA synthesis was studied in an attempt to determine a possible molecular mechanism for age-related biochemical and physiological changes. Initiation of RNA synthesis was determined by incorporation of [gamma-32 P]ATP and of [gamma-32P]GTP into an acid-insoluble product by intact nuclei isolated from livers of Sprague-Dawley CD-strain rats of various ages. When the rats were grouped into young (0.75-9 months) and old (12-30 months) rats, a significant decrease (P less than or equal to 0.001) in incorporation of initiating nucleotides was observed. The rat population was divided into five age groups (0.75-3 months, 4-9 months, 12-18 months, 19-23 months and 30 months) for further analysis of the effect of age on the initiation of RNA synthesis. Analysis of data from these groups indicated a significant trend for an age-related decrease in RNA-synthesis initiation (correlation coefficient = 0.94). Long-term hypophysectomy coupled with minimal hormone-replacement therapy was shown to have a significant effect on the reversal of the age-related decrease in initiation of RNA synthesis. It was observed that initiation of RNA synthesis in nuclei from 19-month-old rats, hypophysectomized at 12 months of age, was closest to that in 3-month-old intact rats and was not significantly different from that in liver nuclei of 0.75-9-month-old intact rats.     

Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Age dependent changes in enzymes involved in macromolecular synthesis in Turbatrix aceti.

The specific activities of elongation factor 1, RNA and DNA polymerase, as well as fructose-1,6-diphosphate aldolase were determined as a function of age in the nematode Turbatrix aceti. It was found that the specific activities of both DNA polymerase and aldolase declined constantly as the animal aged. In contrast, both elongation factor 1 and RNA polymerase showed sharp increases in their specific activities at 5 and 15 days, respectively, before ultimately declining. It was also shown that elongation factor 1, which exists mainly as a high molecular weight species in the young animal, undergoes a conversion to a lower molecular weight species as the organism ages. In addition there is a progressive accumulation of inactive or partially active elongation factor 1 molecules in older animals. The addition of α-tocopherol to the growth medium of these nematodes resulted in an increased life-span as well as alterations in the patterns of the enzyme activities.     

Development of secondary sexual characters in the seabob shrimp Xiphopenaeus kroyeri (Heller 1862) (Crustacea,Decapoda, Penaeidae): a scanning electron microscope study

The development of secondary sexual characters, the petasma, and thelycum growth were studied in Xiphopenaeus kroyeri. In adult females, the thelycum is a single plate and its anterolateral portion is characterized by a reduced hood. The aperture resembles a transverse ridge. In immature stages, the ridge has a space between the plates, which becomes narrower as it reaches the end of development. The female gonopore is ‘comma’ shaped. In adult males, the endopods of the petasma are linked at the dorsomedial margin by a large quantity of cincinnuli. In juveniles, cincinnuli gradually increase in number until they join both endopods. At the end of development the petasma is T-shaped. The male gonopore is C-shaped. The relative growth of the petasma total length versus juvenile body length showed a highly positive allometry, whereas in adults the growth was isometric. For the relationship carapace length versus thelycum width, the juvenile phase of females is characterized by an isometry and the adult phase by a negative allometry.     

Comparative study of physical properties of three suture materials: silk, e-PTFE (Gore-Tex), and PLA/PGA (Vicryl)

The aim of this in-vitro study was to compare the physical properties of three suture materials used in periodontal surgery: silk, expanded polytetrafluoroethylene (e-PTFE), and polylactic acid/polyglycolic and (PLA/PGA). Five physical tests were carried out on each of the three suture materials: strain to failure, tensile strength, knot tensile strength, knot slippage, and capillarity. For each test, 30 samples of each suture material were used. In all cases, the statistical results showed that the e-PTFE and the PLA/PGA threads were superior.