Different routes for integral protein insertion into Ricinus communis protein-body and glyoxysome membranes

Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB antiserum to integral protein-body membrane proteins - anti-G antiserum to integral glyoxysomal membrane proteins - anti-L antiserum to alkaline lipase - ER endoplasmic reticulum - M_r relative molecular mass - mRNA poly(A)-rich messenger RNA - PAGE polyacrylamide gel electrophoresis - poly(A) polyadenylic acid - SDS sodium dodecyl sulphate     

Effects of short-term injection of gonadotrophins on ovarian follicle development in hypogonadal (hpg) mice

Twice daily injections of purified ovine and human FSH were used to investigate the control of ovarian follicle development in hypogonadotrophic hypogonadal (hpg) mice. Treatment for 5 days with doses greater than 3 micrograms resulted in a significant increase in the total number of growing follicles and the development of antral follicles. This was associated with increases in uterine weights and vaginal opening, indicating that steroidogenesis had also been stimulated. Further studies of the effects of combined injections of FSH and LH, linked with morphological analysis of ovarian interstitial cells, suggested that any contribution of background or contaminating LH to the effects of the FSH injections was minimal. It therefore appears that, in mice, FSH alone is capable of stimulating an increase in the initiation of follicle growth, of triggering the development of antral follicles, and supporting ovarian steroidogenesis.     

Growth kinetics as a function of ploidy in diploid, tetraploid, and octaploid smooth muscle cells derived from the normal rat aorta

The smooth muscle cell population in major arteries of humans and experimental animals is heterogeneous with regard to cellular DNA content. A proportion of cells has polyploid DNA content and this proportion increases with normal aging and with hypertension. We have isolated pure populations of rat aortic smooth muscle cells containing 2C, 4C, and 8C DNA content by cloning of cultures of cells previously subjected to flow cytometric cell sorting. Karyologic analysis of these clonal populations revealed them to be pure diploid, tetraploid, and octaploid populations, respectively, containing 2N (= 42), 4N, and 8N chromosomes. Cell attachment area and nuclear size appeared to increase with the level of ploidy. Studies of the proliferative characteristics of the cells revealed that the growth rate and ultimate cell densities achieved decreased as the ploidy level increased. The intrinsic cellular radiosensitivity of these clones did not vary with ploidy. Increased smooth muscle cell ploidy is, therefore, associated with a decreased rate of proliferation. The emergence of smooth muscle cells with polyploid DNA content under normal and pathologic conditions is probably due to mitotic polyploidization without net cell proliferation and may be related to the need for expression of differentiated functions.     

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K_m = 12 micromolar) and coniferaldehyde (K_m = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.     

Adult-young interactions in island and mainland populations of the deermouse Peromyscus maniculatus

Summary The demographic and ecological characteristics of island populations of small mammals have received increasing attention in recent years, but few studies have compared the behavioral characteristics of island populations with those of mainland populations. Behavior is considered an important variable because it is believed by many to be a crucial factor affecting the population dynamics and demography of natural populations. In particular, among many species of rodents, the social behavior of adults towards juveniles is cited as an important factor influencing dispersal patterns and population regulation. The present study compares social interactions between adults and juveniles of island and mainland populations of the deermouse Peromyscus maniculatus, and attempts to relate differences in behavior to the demographic differences between the two populations. Adult mice were trapped on the mainland of British Columbia and on one of the Gulf Islands off the British Columbia coast, and allowed to breed in the laboratory. Male and female juveniles from both populations were then tested with their own parents and with unrelated male and female adults. The results demonstrate that island adults show almost no aggression towards either own or unrelated young. Mainland adults likewise show little aggression towards their own young, but a proportion of the population, consisting of both male and female adults, shows severe aggression towards unrelated juveniles of both sexes. These results suggest four major conclusions: 1) behavior may be the mechanism responsible for the demographic differences reported for these island and mainland populations; 2) female aggression may be a more important factor in deermouse population dynamics than has been previously recognized; 3) since parents show little aggression towards their own young, adult aggression may be a significant factor in juvenile mortality and emigration only after juveniles have initiated dispersal away from their natal sites; and 4) adult aggression controls the number of both male and female juveniles which are recruited into the population.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.