Heterogeneity of hepatic parenchymal enhancement on computed tomography during arterial portography: quantitative analysis of correlation with severity of hepatic fibrosis.

Background/Aims: In patients with chronic liver disease, heterogeneous enhancement of liver parenchyma is often noted on computed tomography during arterial portography (CTAP). We investigated the factors contributing to the heterogeneous enhancement and its relationship with postoperative histopathological findings. Methodology: Eighty-seven patients who had undergone a right lobectomy for liver tumor after CTAP were evaluated. The heterogeneity of hepatic parenchymal enhancement on CTAP was assessed quantitatively using standard deviation of mean CT numbers for five ROIs (S.D.) set in the right hepatic lobe, and comparatively evaluated among three histological groups (liver cirrhosis (LC, n=41), chronic hepatitis (CH, n=33), and normal liver (Normal, n=13)). Severity of fibrosis and degree of splenomegaly (Sp) were taken up as factors contributory to the heterogeneity, and were assessed for correlation with the S.D. Results: The range (mean) of S.D. was LC, 3.07-17.64 (9.10); CH, 1.83-11.12 (6.77); and Normal, 2.06-8.89 (5.64) (Scheffe's F-test: LC vs CH, P<0.0005; LC vs Normal, P<0.0002). The higher fibrosis group exhibited significantly greater S.D. values as compared with the lower fibrosis group (Scheffe's F-test: P<0.00003). Coefficient of correlation between the S.D. and the Sp was 0.295 (P<0.005). Conclusion: There was a fair possibility of LC in patients with heterogeneous enhancement of liver parenchyma on CTAP. The severity of liver fibrosis and the degree of splenomegaly were considered to be factors contributing to the heterogeneous enhancement.

Castleman's disease of the spleen: CT, MR imaging and angiographic findings.

Since Castleman and Towne [Castleman and Towne, Hyperplasia of mediastinal lymph nodes, New Engl. J. Med. 250 (1954), 26-30] first described hyperplasia of the mediastinal lymph nodes in 1954, many cases of Castleman's disease have been reported. Lesions originating in the spleen arc extremely rare, and we here describe the imaging appearances for such a case, and discuss with a brief review of the literature.

Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export.

Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

Interactions between a nuclear transporter and a subset of nuclear pore complex proteins depend on Ran GTPase.

Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with importin-alpha and -beta (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of interaction with nucleoporins. Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, the nucleoporin association is lost. A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins. These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins. These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC.

A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export.

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin beta superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.

Protein translocation functions of Escherichia coli SecY: in vitro characterization of cold-sensitive secY mutants.

Protein translocation across the plasma membrane of E coli is facilitated by Sec factors, including the membrane-embedded SecYEG subunit and the SecA ATPase. Although there is complete agreement that SecA is essential for protein translocation, some publications question the essentialness of SecY. We previously isolated a number of cold-sensitive mutants of secY and characterized their in vivo phenotypes. In this study, we characterized membrane vesicles prepared from these mutants with respect to their in vitro activities to support protein translocation and to activate the SecA ATPase. These studies revealed several single amino acid alterations that abolish these in vitro activities of membrane vesicles. In particular, several mutations in the two most carboxy-terminal cytoplasmic domains of SecY prevented SecA from functioning as the translocation ATPase. A number of mutants showed strong correlations between in vivo protein export ability, in vitro translocation activity and in vitro translocation ATPase activity, substantiating the importance of SecY in vivo and in vitro. A few other mutants were affected in only one or two aspects of these properties, suggesting that they were differentially affected in some substeps of translocation. These results provide further evidence that SecY has vital roles in protein translocation, in which the 'motor' function of SecA and the 'channel' function of SecYEG should be coordinated.

Yrb2p is a nuclear protein that interacts with Prp20p, a yeast Rcc1 homologue.

A conserved family of Ran binding proteins (RBPs) has been defined by their ability to bind to the Ran GTPase and the presence of a common region of approximately 100 amino acids (the Ran binding domain). The yeast Saccharomyces cerevisiae genome predicts only three proteins with canonical Ran binding domains. Mutation of one of these, YRB1, results in defects in transport of macromolecules across the nuclear envelope (Schlenstedt, G., Wong, D. H., Koepp, D. M., and Silver, P. A. (1995) EMBO J. 14, 5367-5378). The second one, encoded by YRB2, is a 327-amino acid protein with a Ran binding domain at its C terminus and an internal cluster of FXFG and FG repeats conserved in nucleoporins. Yrb2p is located inside the nucleus, and this localization relies on the N terminus. Results of both genetic and biochemical analyses show interactions of Yrb2p with the Ran nucleotide exchanger Prp20p/Rcc1. Yrb2p binding to Gsp1p (yeast Ran) as well as to a novel 150-kDa GTP-binding protein is also detected. The Ran binding domain of Yrb2p is essential for function and for its association with Prp20p and the GTP-binding proteins. Taken together, we suggest that Yrb2p may play a role in the Ran GTPase cycle distinct from nuclear transport.

Genetic analysis of macromolecular transport across the nuclear envelope.

Numerous factors that promote movement of macromolecules in and out of the nucleus have now been identified. These include both soluble cytoplasmic and nucleoplasmic proteins and proteins of the nuclear pore complex (NPC). Genetic analyses of the nuclear transport process in the model organism, the budding yeast Saccharomyces cerevisiae, have revealed remarkable conservation of all of these factors. In addition, important clues as to how these factors promote the unique bidirectional movement across the NPC have emerged from studies of yeast. We summarize the characterization and genetic interactions of the soluble transport factors and present data to illustrate how genetic experiments can be used to further define the import and export pathways.

Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli.

A mutant form of SecY, SecY-d1, was previously suggested to sequester a component(s) of the protein translocator complex. Its synthesis from a plasmid leads to interference with protein export in Escherichia coli. SecE is a target of this sequestration, and its overproduction cancels the export interference. We now report that overexpression of another gene, termed syd, also suppresses secY-d1. The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino-terminal sequence. Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane. SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available. Overproduction of Syd was found to stabilize oversynthesized SecY. However, Syd cannot stabilize the SecY-d1 form of SecY. Thus, in the presence of both secY+ and secY-d1, Syd increases the effective SecY+/SecY-d1 ratio in the cell and cancels the dominant interference by the latter. We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened. These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY. Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.

A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex.

An approach to identifying the interaction site of multicomponent protein assembly has been applied to the membrane-bound SecY-SecE complex, which mediates protein export across the Escherichia coli cytoplasmic membrane. A dominant negative secY allele, secY-d1, inactivates SecY but preserves its ability to interact with SecE. Thus, the mutant protein sequesters SecE in an inactive complex. Second site mutations that disrupt the SecE binding site will suppress the export interference. We introduced insertion/deletion mutations that intragenically suppressed secY-d1. After eliminating knock-out mutations by virtue of the expression of a LacZ alpha sequence that had been attached to the C terminus, we obtained a striking clustering of mutations in cytoplasmic domain 4. On the basis of this result, the secY24 (Ts) substitution mutation in this domain was examined for its effects on interaction with SecE. It indeed suppressed secY-d1. Although the instability associated with excess SecY can be alleviated by overproduction of SecE, the secY24 mutant protein was not stabilized by SecE. The basal-level SecY24 protein was also destabilized at 42 degrees C. SecE was coimmunoprecipitated with SecY+ but not with the SecY24 protein. These results indicate that the secY24 mutation weakens SecY's interaction with SecE. Taken together, we propose that cytoplasmic domain 4 is important for the association between SecY and SecE.

Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes.

A number of secY mutants of Escherichia coli showing protein export defects were isolated by a combination of localized mutagenesis and secA-lacZ screening. Most of them were cold sensitive and contained single base substitutions in secY leading to amino acid replacements in various parts of the SecY protein, mainly in the cytoplasmic and the transmembrane domains. A temperature-sensitive mutant with an export defect had the same base substitution as secY24, which was characterized previously. Many cold-sensitive secY mutants exhibited rapid responses to temperature lowering but their apparent defects varied at the permissive temperature. Others exhibited delayed responses to the temperature shift. Some secY mutations, including secY39, interfered with protein export when expressed from a multicopy plasmid, even in the presence of wild-type secY on the chromosome. Such "dominant negative" mutations, including secY-d1, which was studied previously, were all located in either cytoplasmic domain 5 or 6, which is consistent with our previous proposal that the C-terminal region of SecY is important for its function as a protein translocator. We also studied the phenotypes of strains in which one of the secY mutations was combined with the components of the secD operon. Overexpression of secD partially suppressed the secY39 mutation, while overexpression of secF exacerbated the export defects of secY122 and secY125 mutations. Overexpression of "yajC", located within the secD operon, suppressed secY-d1. Although yajC itself proved to be dispensable, its disruption impaired the growth of the secY39 mutant at 42 degrees C. These observations suggest that SecY interacts with SecD, SecF, and the product of yajC.

Determinants of the quantity of the stable SecY complex in the Escherichia coli cell.

While SecY in wild-type Escherichia coli cells is stable and is complexed with other proteins within the membrane, moderately overexpressed and presumably uncomplexed SecY was degraded with a half-life of 2 min. The fact that the amount of stable SecY is strictly regulated by the degradation of excess SecY was demonstrated by competitive entry of the SecY+ protein and a SecY-LacZ alpha fusion protein into the stable pool. Simultaneous overexpression of SecE led to complete stabilization of excess SecY. Overproduced SecD and SecF did not affect the stability of SecY, but plasmids carrying ORF12 located within the secD-secF operon partially stabilized this protein. In contrast, mutational reduction of the SecE content (but not the ORF12 content) led to the appearance of two populations of newly synthesized SecY molecules, one that was immediately degraded and one that was completely stable. Thus, the E. coli cell is equipped with a system that eliminates SecY unless it is complexed with SecE, a limiting partner of SecY. Our observations implied that in wild-type cells, SecY and SecE rapidly associate with each other and remain complexed.

Inhibition of in utero galactose-induced cataract development by an aldose reductase inhibitor in rat--electron microscopic study.

The effect of an aldose reductase inhibitor (ARI) on the development of in utero galactose-induced cataract was evaluated by following the morphological changes as seen by light and transmission electron microscope. Pregnant rats were fed a 30% galactose diet until day 17, 18, 19 or 20 of gestation and then given a galactose diet containing ARI. The lenses of newborn rats when mothers were given an ARI diet after day 17, 18 or 19 of gestation showed no morphological change. On the other hand, in the lenses from newborn rats when mothers were given an ARI diet after day 20 of gestation, 30% of those tested showed small vacuoles limited to the posterior cortex, while 70% showed normal structure. When mothers were given a galactose diet during the entire term of pregnancy all lenses of newborn rats showed extensive vacuole formation. ARI was confirmed to completely inhibit congenital galactose cataracts in rats when it was given to the mother rat after day 19 of gestation (ie, on the last 3 days before delivery).

Effect of pregnancy on development of galactose-induced cataract in rat.

The effect of pregnancy on the development of galactose-induced cataract was studied in rats. Eight-week-old rats were fed a 50% galactose diet for 4 weeks. The rats were divided into three groups: nonpregnant females, pregnant females, and males. All eyes were examined by slit-lamp biomicroscope at 11 and 12 weeks of age, and the lenses were morphologically examined at 12 weeks. Clinically, the cataract showed a less advanced stage in the eyes of pregnant rats than in the eyes of the other two groups. Morphologically, the lenses of pregnant rats retained their normal structure. However, the lenses of the other two groups showed extensive damage. The plasma galactose levels and galactitol levels in lenses showed no significant difference among the three groups. Pregnancy was proved to inhibit the development of galactose cataract.

Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation.

The Escherichia coli gene ssyB was cloned and sequenced. The ssyB63 (Cs) mutation is an insertion mutation in nusB, while the nusB5 (Cs) mutation suppresses secY24, indicating that inactivation of nusB causes cold-sensitive cell growth as well as phenotypic suppression of secY24. The correct map position of nusB is 9.5 min rather than 11 min as previously assigned. It is located at the distal end of an operon that contains a gene showing significant homology with a Bacillus subtilis gene involved in riboflavin biosynthesis.

SecY variants that interfere with Escherichia coli protein export in the presence of normal secY.

As an approach for studying how SecY, an integral membrane protein translocation factor of Escherichia coli, interacts with other protein molecules, we isolated a dominant negative mutation, secY-d1, of the gene carried on a plasmid. The mutant plasmid severely inhibited export of maltose-binding protein and less severely of OmpA, when introduced into sec+ cells. It inhibited growth of secY and secE mutant cells, but not of secA and secD mutant cells or wild-type cells. The mutation deletes three amino acids that should be located at the interface of cytoplasmic domain 5 and transmembrane segment 9. We also found that some SecY-PhoA fusion proteins that lacked carboxy-terminal portions of SecY but retain a region from periplasmic domain 3 to transmembrane segment 7 were inhibitory to protein export. We suggest that these SecY variants are severely defective in catalytic function of SecY, which requires cytoplasmic domain 5 and its carboxy-terminal side, but retain the ability to associate with other molecules of the protein export machinery, which requires the central portion of SecY; they probably exert the 'dominant negative' effects by competing with normal SecY for the formation of active Sec complex. These observations should provide a basis for further genetic analysis of the Sec protein complex in the membrane.

Does protein secretion activity vary during the cell cycle of Escherichia coli?

It is not known whether the activity of the Escherichia coli protein export system changes during the division cycle. To address and answer this question, we took two approaches. First, we pulse-labeled a random culture and size-fractionated the labeled cells in the presence of inhibitors of secretion. Second, we pulse-labeled synchronously growing cells at different phases in the cell cycle. In the latter experiment, we used a new method of synchronization in which a random culture was simply filtered through glass fiber filters. In both cases, the proportions of unprocessed precursor molecules were measured by immunoprecipitation and gel electrophoresis for some representative periplasmic and outer membrane proteins. Within the sensitivity limits of these methods, we could not detect any significant variation in the precursor labeling for cells of different ages. Thus, E. coli cells appear to secrete proteins continuously throughout the division cycle.