Proalbumin is processed to serum albumin in COS-1 cells transfected with cDNA for rat albumin

Synthesis and processing of rat albumin were investigated in COS-1 cells transiently expressing rat albumin. Analysis using isoelectric focusing revealed that serum-type albumin, which is indistinguishable from the counterpart isolated from rat hepatocyte cuture medium, was secreted from the transfected COS-1 cells, indicating that proalbumin is effectively converted into serum albumin in the COS-1 cells, if not completely. Furthermore methylamine was found to cause the diminution of serum albumin released from the cells, substantiating that the proteolytical conversion of proalbumin occurs in the Golgi complex before discharge from the COS-1 cells.     

Disparate effects of monensin and colchicine on intracellular processing of secretory proteins in cultured rat hepatocytes

We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, alpha 1-protease inhibitor and alpha 2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, alpha 1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51 000, and then processed to two endoglycosidase-H-resistant forms having Mr 51 000 and 56 000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for alpha 2u-globulin. In the cells treated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed alpha 1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized alpha 1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51 000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant alpha 1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

5'-nucleotidase from the electric ray electric lobe. Primary structure and relation to mammalian and procaryotic enzymes.

A cDNA encoding a 5'-nucleotidase was identified by screening a lambda gt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63,833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5'-nucleotidase shares 61% amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5'-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5'-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5-7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5'-nucleotidase, 3'-nucleotidase or phosphodiesterase activity. 5'-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5'-nucleotidase from multifunctional nucleotide hydrolases is discussed.     

Selective processing of proalbumin determined by site-specific mutagenesis

Rat proalbumin is cleaved at the dibasic pair Arg-Arg and converted into a mature form with Glu at the NH2 terminus. In the present study site-directed mutagenesis of the albumin cDNA was designed to generate proalbumin variants in which Glu1 was substituted with various amino acid residues. The expression plasmids constructed were transfected into COS-1 cells, and the intracellular processing of proalbumins expressed was examined by labeling experiments. Substitution of Glu1----Ser allowed the expressed proalbumin to be processed as observed for the wild-type precursor. However, replacement of Glu1 with a hydrophobic residue (Val, Leu or Ile) resulted in no processing of proalbumin, despite retaining the same cleavage signal Arg-Arg as above. The results indicate that the residue at position 1 adjacent to the dibasic pair is also important for recognition by the proalbumin-processing enzyme.     

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis.

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.     

Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation.

The binding of [${}^3\text{H}$]kanamycin to $\text{E. coli}$ ribosomes and ribosomal subunits was studied by equilibrium dialysis and Millipore filter methods. The 70S ribosome bound ca. two molecules up to the antibiotic concentration of 10 uM, and more at higher concentrations. Each ribosomal subunit was observed to possess one major binding site, and the affinity of the small ribosomal subunit was greater than that of the large subunit. The binding of [${}^3\text{H}$]kanamycin to ribosomes and ribosomal subunits was reversed by neomycin or gentamicin, but not by streptomycin and chloramphenicol. Kanamycin, neomycin and gentamicin interfered with the binding of [${}^{14}\text{C}$] tuberactinomycin O. Translocation of N-Ac-Phe-tRNA was markedly inhibited by kanamycin, neomycin or gentamicin, but not by streptomycin.     

Does the rate of ribosomal RNA synthesis vary depending on the number of nucleoli in a nucleus?

Cultured kidney cells of Xenopus laevis were pulse-labeled with [³H]uridine for 10, 20 and 30 min during their logarithmic growth phase and then processed for autoradiography. The labeled cells were assigned into two categories, one- and two-nucleolated cells, and the rate of ribosomal RNA (rRNA) synthesis was measured by counting the number of grains in nucleoli. The results obtained revealed that a two-nucleolated cell incorporated significantly much more radioactivity into its nucleoli than did a one-nucleolated partner for all the periods examined. Cells of these different nucleolar types, however, contained essentially the same amount of rDNA (DNA complementary to rRNA) as estimated by in situ hybridization with [¹²⁵I]rRNA.Although it remains to be proved that the observed increase in incorporation represents the increased rate of rRNA synthesis in two-nucleolated cells, the present findings seem to be very interesting, since they might indicate that the activity of rRNA genes is in some way regulated or affected by their spatial relationship in a cell nucleus.     

The relationship between consumption of tyrosine and phenylalanine as precursors of catecholamine at breakfast and the circadian typology and mental health in Japanese infants aged 2 to 5 years

Background

This study aims to examine the relationship between tyrosine and phenylalanine intake at breakfast as precursors of dopamine, and scores on the Torsvall-Åkerstedt Diurnal Type Scale and of mental health in Japanese infants aged 2 to 5 years.

Results

An integrated questionnaire was administered to parents of 1,367 infants attending one of ten nursery schools governed by Kochi City or a kindergarten affiliated with the Faculty of Education at Kochi University (775 answers for analysis: 56.7%) in May and June 2008. Questionnaires included the Torsvall-Åkerstedt Diurnal Type Scale and questions on sleep habits (onset, offset, quality, quantity, and so on), meal habits (content and regularity of timing), and mental health (depressive states). Amount of tyrosine and phenylalanine intake was calculated based on a breakfast content questionnaire and data on the components of amino acids in foods. Infants who ingested more than 800 mg of tyrosine or phenylalanine at breakfast per meal were more morning-type than those who ingested less than 800 mg (ANOVA: P= 0.005). However, this relationship disappeared in the ANCOVA analysis (with the covariance of tryptophan intake, P= 0.894). Infants who ingested more than 800 mg of the two amino acids at breakfast showed significantly higher mental health scores (lower frequency of depressive states) than those who ingested less than 800 mg (ANOVA: P = 0.004). This relationship remained significant when ANCOVA analysis was performed with the covariance of tryptophan (ANCOVA: P= 0.017).

Conclusions

These results suggest that tyrosine and phenylalanine ingested at breakfast are not related with circadian phase, but are relate with mental health in infants.